Methods for epicardial differentiation of human pluripotent stem cells

ABSTRACT

Methods for generating high-yield, high-purity epicardial cells are described. Wnt/β-catenin signaling is first activated in human cardiac progenitor cells, by, for example, inhibiting Gsk-3 to induce differentiation into epicardial cells. Methods for long-term in vitro maintenance of human cardiac progenitor cell-derived epicardial cells and method comprising chemically defined, xeno-free, and albumin-free culture conditions are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/143,359, filed Apr. 6, 2015, which is incorporated herein byreference as if set forth in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under EB007534 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND

The mammalian heart, the first functional organ in the developingvertebrate embryo, includes three distinct structures—the epicardium,myocardium, and endocardium. The epicardium, the outermost layer of theheart, contributes both multi-lineage descendants and important trophicsignals to the myocardium and coronary vessels. The epicardium developsfrom the proepicardium, a mass of coelomic progenitors located at thevenous pole of the embryonic heart. Proepicardium cells attach to andspread over the myocardium to form the primitive epicardial epithelium.During cardiogenesis, epicardial cells undergo epithelial-to-mesenchymaltransition (EMT) to give rise to a population of epicardium-derivedcells, which in turn invade the heart and progressively differentiateinto various cell types, including cells of coronary blood vessels andcardiac interstitial cells.

The epicardium contributes both multi-lineage descendants and paracrinefactors during cardiac repair, underscoring their potentials forregenerative medicine (Lepilina et al., Cell. 127:607-619 (2006);Kikuchi et al., Dev. Cell. 20:397-404 (2011); Zhou et al., J. Clin.Invest. 121:1894-1904 (2011); Zhou & Pu, J. Cell. Mol. Med. 15:2781-2783(2012)). Understanding the molecular mechanisms that control thespecification of epicardial lineages from naïve progenitor cells isfundamental to elucidating the regulatory mechanisms underlying bothhuman heart development and cardiovascular diseases. Because of theirdevelopmental importance and therapeutic potential, epicardial cells andepicardium-derived cells present a tractable resident progenitor sourceto restore a functional vasculature, to maintain cardiomyocyte survival,and to repair damaged heart tissue. Accordingly, there is a need in theart for efficient and cost-effective protocols for generating functionalepicardial cells under chemically defined culture conditions and in theabsence of certain growth factors previously thought to be an essentialpart of directed epicardial differentiation.

BRIEF SUMMARY

The invention relates generally to methods for cardiac induction inhuman pluripotent stem cells (hPSCs) and, more particularly, to methodsfor generating from hPSCs populations of functional epicardial cellsunder chemically-defined, albumin-free conditions and for long-termmaintenance of such hPSC-derived epicardial cell populations.

In one aspect, provided herein is a method for generating a populationof epicardial cells from human pluripotent stem cells. Generally, themethod comprises culturing human cardiac progenitor cells differentiatedfrom human pluripotent stem cells in a chemically defined, albumin-freeculture medium that comprises an activator of Wnt/β-catenin signaling,whereby a cell population comprising human epicardial cells is obtained.The human cardiac progenitor cells express one or more of Isl1, Nkx2.5,and Flk-1. In some cases, at least 95% of cells of the cell populationare epicardial cells positive for expression of Wilms' tumor suppressorprotein (WT1). In some cases, the chemically defined culture medium doesnot comprise Bone Morphogenetic Protein 4 (BMP4). The activator ofWnt/β-catenin signaling can be a Gsk3 inhibitor. The Gsk3 inhibitor canbe a small molecule selected from the group consisting of CHIR99021,CHIR98014, BIO-acetoxime, BIO, LiCl, SB216763, SB415286, AR A014418,1-Azakenpaullone, and Bis-7-indolylmaleimide. The Gsk3 inhibitor can beCHIR99021 and is present in a concentration of about 0.2 μM to about 9μM.

The human cardiac progenitor cells can be obtained by a methodcomprising (i) culturing human pluripotent stem cells in a chemicallydefined culture medium comprising an activator of Wnt/β-cateninsignaling to obtain a first cell population comprising mesodermal cellspositive for expression of Brachyury/T; and (ii) culturing the firstcell population in a chemically defined culture medium that comprises aninhibitor of Wnt/β-catenin signaling, whereby a cell populationcomprising human cardiac progenitor cells is obtained. In some cases,the human cardiac progenitor cells express one or more of Isl1, Nkx2.5,and Flk-1. The activator of Wnt/β-catenin signaling can be a Gsk3inhibitor. The Gsk3 inhibitor can be a small molecule selected from thegroup consisting of CHIR99021, CHIR98014, BIO-acetoxime, BIO, LiCl,SB216763, SB415286, AR A014418, 1-Azakenpaullone, andBis-7-indolylmaleimide. The inhibitor of Wnt/β-catenin signaling can beselected from the group consisting of a small molecule that stabilizesaxin and stimulates β-catenin degradation, an inhibitor of porcupine, anantibody that blocks activation of a Wnt ligand receptor, an antibodythat binds to one or more Wnt ligand family members, and a short hairpininterfering RNA (shRNA) for β-catenin in the first cell population. Thesmall molecule that stimulates β-catenin degradation and stabilizes axincan be XAV939. The porcupine inhibitor can be selected from the groupconsisting of IWP2 and IWP4, or a combination thereof. The porcupineinhibitor can be present in a concentration of about 1 μM to about 4 μM.In some cases, no cell separation or selection step is used to obtainthe cell population comprising epicardial cells.

In another aspect, provided herein is a method for long-term in vitromaintenance of self-renewing human epicardial cells, the methodcomprising culturing human cardiac progenitor cells differentiated fromhuman pluripotent stem cells in a chemically defined, albumin-freeculture medium that comprises an activator of Wnt/β-catenin signaling,whereby a cell population comprising human epicardial cells is obtained;and culturing the cell population comprising human epicardial cells inthe presence of an inhibitor of TGFβ signaling, whereby the humanepicardial cells are self-renewing for at least 25 population doublings,are not immortalized, and maintain the ability to undergoepithelial-to-mesenchymal transition (EMT).

In a further aspect, provided herein is a kit for differentiating humanpluripotent stem cells into epicardial cells. Generally, the kitcomprises (i) a culture medium suitable for differentiating humancardiac progenitor cells into epicardial cells; (ii) an agent thatactivates Wnt signaling in human cardiac progenitor cells; and (iii)instructions describing a method for generating human epicardial cells,the method employing the culture medium and the agent. The kit canfurther comprise instructions describing methods for long-term in vitromaintenance of the human epicardial cells, where the method employs aculture medium and an agent that supports long-term maintenance of suchepicardial cells.

These and other features, objects, and advantages of the presentinvention will become better understood from the description thatfollows. In the description, reference is made to the accompanyingdrawings, which form a part hereof and in which there is shown by way ofillustration, not limitation, embodiments of the invention. Thedescription of preferred embodiments is not intended to limit theinvention to cover all modifications, equivalents and alternatives.Reference should therefore be made to the claims recited herein forinterpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood and features, aspectsand advantages other than those set forth above will become apparentwhen consideration is given to the following detailed descriptionthereof. Such detailed description makes reference to the followingdrawings, wherein:

FIGS. 1A-C demonstrate that temporal Wnt signaling modulation issufficient for epicardial linage specification from hPSC-derived cardiacprogenitors. A schematic representation of protocols used todifferentiate Nkx2.5⁺Isl1⁺ cardiac progenitors toward the epicardiallineages is presented in (A). H13 hESC-derived day 12 cultures weresubjected to flow cytometry analysis (B) and immunostaining analysis (C)for Wilms' tumor suppressor protein 1 (WT1) and cTnT. Scale bars, 100μm. Data are represented as mean±SEM of at least three independentreplicates.

FIGS. 2A-E illustrate generation of WT1-2A-eGFP knock-in hPSC linesusing Cas9 nuclease. (A) Schematic diagram of the targeting strategy atthe stop codon of the WT1 locus. Vertical arrows indicate sgRNA1 andsgRNA2 targeting sites. Red and blue horizontal arrows indicate PCRgenotyping primers for assaying WT1 locus targeting and homozygosity,respectively. (B) PCR genotyping of hESC clones after puromycinselection and the expected PCR product for correctly targeted WT1 locusis ˜3 kbp (red arrows) with an efficiency of 21/44. Correctly targetedclones underwent a further homozygosity assay. Clones with the PCRproducts of about 200 bp are heterozygous (blue arrow), and those cloneswithout PCR products are homozygous. (C) PCR genotyping of hESC clonesafter TAT-Cre mediated excision of the PGK-Puro cassette. Clones withthe PCR products of about 1 kbp are PGK-Puro free, and those with ˜3 kbpcontain PGK-Puro. (D) Live cell flow analysis of GFP+ cells at day 0,day 10 and day 12 during CHIR treatment of WT1-2A-eGFP knock-in ES03.(E) Phase contrast images and corresponding eGFP fluorescent images ofWT1-2A-eGFP hPSC-derived epicardial cells after excision of the PGK-Purocassette. Scale bars, 100 μm.

FIGS. 3A-E demonstrate use of chemically-defined, albumin-freeconditions to generate WT1+ epicardial cells. (A) Schematic of theprotocol for chemically-defined differentiation of hPSC-derived cardiacprogenitors to WT1+ epicardial cells via GSK3 inhibition. (B) H13hESC-derived day 6 cardiac progenitor cells were cultured as illustratedin (A) in LaSR basal medium with indicated CHIR99021 (CH)concentrations. At day 12, cells were analyzed for WT1 expression byflow cytometry. (C) Representative flow analysis of WT1-eGFP knockinES03-derived epicardial cells with indicated CHIR concentration. (D) H13hESC-derived day 6 cardiac progenitor cells were cultured as illustratedin (A) with the indicated day 6 cell seeding density in LaSR basalmedium. At day 12, cells were analyzed for WT1 expression by flowcytometry. (E) H13 hESC-derived day 6 cardiac progenitor cells wereseeded at a density of 0.06 million cells/cm² and cultured asillustrated in (A) with 3 μM CHIR99021 in the indicated basal medium. Atday 12, cells were analyzed for WT1 expression by flow cytometry. Dataare represented as mean±SEM of at least three independent replicates.

FIGS. 4A-C demonstrate β-catenin-dependent epicardial celldifferentiation from hPSC-derived cardiac progenitors. (A) Day 6 H13hESC-derived cardiac progenitor cells were seeded at a density of 0.06million cells/cm², differentiated in LaSR basal medium with DMSO, 0.3 μMCHIR98014, 0.3 μM BIO-acetoxime or 3 μM CHIR99021 from day 7 to day 9,and subjected to flow cytometry analysis of WT1 expression at day 12. #p<0.005, DMSO versus CHIR98014, BIO-acetoxime, and CHIR99021; t test.(B) 19-9-11 iscramble and 19-9-11 ishcat-1 iPSCs were cultured in E8medium with or without 2 μg/ml doxycycline (dox). After 3 days, mRNA wascollected and CTNNB1 expression evaluated by qPCR. *p<0.005, ishcat-1with dox versus ishcat-1 without dox or iscramble (w/o dox); t test. (C)19-9-11 ishcat-1 iPSC-derived day 6 cardiac progenitor cells weredifferentiated as illustrated in FIG. 3A with 2 μg/ml doxycyclineaddition at the indicated times. At day 12, cells were analyzed for WT1+and cTnT+ expression by flow cytometry. Data are represented as mean±SEMof at least three independent replicates.

FIGS. 5A-E present molecular analysis of hPSC-derived epicardial cellsobtained under chemically-defined, albumin-free conditions. (A)Schematic of the optimized protocol for differentiation of hPSCs toepicardial cells in RPMI medium. (B-D) H13 hESC-derived cardiacprogenitors were differentiated as illustrated in (A). Gene expressionwas assessed by quantitative RT-PCR (B). Data are represented asmean±SEM of at least three independent replicates. (C) At different timepoints, WT1 and TBX18 expression was assessed by western blot. (D) Atday 12, immunostaining for TBX18 and TCF21 was performed. Scale bars, 50μm. (E) Representative phase contrast microscopy and fluorescenceimmunostaining for WT1, ZO1, and β-catenin of day 12 pro-epicardium(Pro-Epi) and day 18 epicardium (Epi). Scale bars, 100 μm.

FIGS. 6A-D demonstrate that hPSC-derived epicardial cells undergo EMT inresponse to bFGF and TGFβ1 treatment, yielding epicardium-derived cellsthat display characteristics of fibroblasts and vascular smooth musclecells. (A) Schematic of the protocols used for the EMT induction of H13hESC-derived epicardial cells with 10 ng/mL bFGF and 5 ng/mL TGFβ1. (B)At day 18, phase contrast images displaying cell morphology andfluorescence images showing the presence of WT1, ZO1, α-SMA and TCF21proteins in Epi-derived cultures. Scale bars, 100 μm. (C) qPCR analysisof EMT related genes SNAIL2, CDH2 and CDH1 and (D) immunostaininganalysis of E-cadherin expression after the indicated bFGF and TGFβ1treatments. Scale bars, 50 μm.

FIGS. 7A-D demonstrate long-term expansion of hPSC-derived epicardialcells. (A-B) H13 hESC-derived day 18 epicardial cells were seeded at adensity of 0.05 million cells/cm² and treated with the indicated smallmolecules for 3 days (concentrations provided in Table 3). At day 4,representative phase contrast microscopy and fluorescence immunostainingfor WT1, ZO1 and α-SMA (A), and the total cell numbers were assessed(B). Scale bars, 100 μm. (C-D) H13 hESC-derived epicardial cells werepassaged and counted every four days in the absence or presence of theindicated TGFβ inhibitors: 0.5 μM A83-01 or 2 μM SB431542. Thepopulation doublings were calculated and shown in (C), and day 48cultures were subjected to flow analysis of WT1 and Ki67 expression (D).

FIGS. 8A-E show that hPSC-derived epicardial cells were similar toprimary epicardial cells. (A) Hierarchical clustering analysis ofRNA-seq expression data of human pluripotent stem cells (hPSCs),hPSC-derived endoderm (Endo), hPSC-derived ectoderm (Ecto), hPSC-derivedmesoderm (Mes), CMs, epicardial cells (Epi) derived from human stem celllines H9, ES03, and 19-9-11, and primary epicardial cells (donor 9605,9633, 9634, and 9635). (B) Before surgery, ES03-eGFP cells weredifferentiated as illustrated in FIG. 5A, cultured for 5 passages in aA83-01-containing medium, and subjected to flow cytometry analysis forWT1 and GFP expression. Histogram distribution of depth of invasion (C)of eGFP-positive cells in 2-, 6-, and 12-day sections are shown. Scalebars, 100 μm. After 12 days, hearts were harvested and representativesmooth muscle actin (SMA) and human-specific mitochondria (Mito) dualstain (D) of cross-sections of the heart are shown. Arrows denote thescaffold. Scale bar, 100 μm. (E) Model highlighting the specification ofhPSCs to epicardial lineages by stage-specific modulation of canonicalWNT signaling and the long-term maintenance of hPSC-derived epicardialcells via inhibition of TGFβ signaling.

FIGS. 9A-C present albumin-free differentiation of NKX2.5+ISL1+FLK-1+cardiac progenitors from hPSCs by small molecule modulation of Wntsignaling. (A) Schematic of the protocol for defined, albumin-freedifferentiation of hPSCs to cardiac progenitors in RPMI medium. (B) H13hESCs were differentiated as illustrated in (A), re-passaged on day 6and immunostained for indicated markers on day 7. Scale bars, 100 μm.(C) H13 hESCs were differentiated as illustrated in (A) anddevelopmental gene expression was assessed by quantitative RT-PCR atindicated time points. Data are represented as mean±SEM of at leastthree independent replicates.

FIG. 10 presents flow cytometry analysis of cTnT and WT1 expression inday 12 H13 hESC cultures differentiated as shown in FIG. 1A in thepresence or absence of 2 μM CHIR99021 and 10 ng/mL BMP4.

FIG. 11 demonstrates 19-9-11 ishcat-1 iPSC-derived day 6 cardiacprogenitor cells differentiated as illustrated in FIG. 3A with 2 μg/mldoxycycline addition at the indicated times. Representative flowcytometry analysis of cTnT and WT1 expression with the indicated doxtreatments were shown.

FIGS. 12A-C demonstrate epicardial cells maturation following passage ata low density in chemically-defined medium. H13 hESC-derived day 6cardiac progenitor cells were seeded at a density of 0.06 million cellsper cm² as illustrated in FIG. 5A in RPMI/Vc/Ins medium with 3 μMCHIR99021 from day 7 to day 9. At different time points, TCF21 andALDH1A2 (A) expression was assessed by western blot. After passage at adensity of 0.05 million cells per cm², differentiated cultures weresubjected to qPCR (B) and immunostaining (C) analysis of ALDH1A2. Scalebars, 50 μm. Data are represented as mean±SEM of at least threeindependent replicates.

FIGS. 13A-B show differentiation of multiple hESC and iPSC lines toepicardial cells. Epicardial cells were generated as described in FIG.5A from different hPSC lines: hESC H9, hESC ES03, iPSC 19-9-7. Day 12pro-epicardial cells were subjected to flow cytometry analysis of WT1expression (A), and representative contrast and immunostaining images ofWT1, ZO1 and β-catenin of post-passage day 18 epicardial cells are shownin B. Scale bars, 50 μm.

FIG. 14 demonstrates that epicardial cells underwent EMT in response tobFGF and TGFβ1 treatment. Immunostaining analysis of indicatedfibroblast, smooth muscle and endothelial cell markers in LaSR basalmedium with bFGF and bFGF+TGFβ, and EGM-2 medium, respectively. Data arerepresented as mean±SEM of at least three independent replicates.

FIGS. 15A-D present data from long-term maintenance of 19-9-11iPSC-derived epicardial cells. hPSCs were differentiated to epicardialcells as illustrated in FIG. 5A, passaged and counted every four days inthe presence of the indicated TGFβ inhibitors: 0.5 μM A83-01 or 2 MSB431542. The population doublings were calculated and shown in (A), andday 48 cultures were subjected for flow analysis of ALDH1A2 (B) and Ki67(C) expression. (D) Representative phase contrast and immunostainingimages of WT1, ZO1 and β-catenin in day 48 epicardial cells treated withA83-01 are shown. Scale bars, 50 μm.

FIGS. 16A-G demonstrate that hPSC-derived epicardial cells arefunctionally similar to primary epicardial cells both in vitro and invivo. (A) Hierarchical clustering of the top 50 significantly enrichedpathways in indicated cell types. The color bar indicates the absolutenormalized enrichment score (NES) for the enriched pathways. (B) 3Dscores plot of first 3 principal components (PCs) from the PCA. Theellipses show the 95% confidence limit and each data point correspondsto different biological samples. Black arrows show the developmenttransition from hPSCs to mesoderm, from which CMs and epicardial cellsarise. (C) Venn diagram showing the number of pathways which areenriched in different cell types (relative to hPSCs). Top 150significantly enriched pathways (p<0.05), ranked by absolute NES foreach cell type was used for analysis. (D) The cardiac fibroblast-derivedextracellular matrix (CF-ECM) patch seeded with cells was photographedat time of placement and 12 days after transplantation. Black arrowsdenote the sutures for MI and white arrows denote the CF-ECM scaffold.Scale bars, 1 cm. Representative photomicrographs (E) of eGFP-positivecells in 2-, 6-, and 12-day sections are shown. Scale bars, 100 μm.After 12 days, hearts were harvested and representative hematoxilin andeosin stain (HE stain) (F), vimentin (VIM) and calponin (G) stain ofcross-sections of the heart are shown. Arrows denote the scaffold. Scalebar, 100 μm.

While the present invention is susceptible to various modifications andalternative forms, exemplary embodiments thereof are shown by way ofexample in the drawings and are herein described in detail. It should beunderstood, however, that the description of exemplary embodiments isnot intended to limit the invention to the particular forms disclosed,but on the contrary, the intention is to cover all modifications,equivalents and alternatives falling within the spirit and scope of theinvention as defined by the appended claims.

DETAILED DESCRIPTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though set forth in their entirety in the presentapplication.

The methods and compositions provided herein are based at least in parton the Inventors' discovery that differentiation stage-specificmodulation of canonical Wnt signaling is sufficient for efficientepicardial induction from human pluripotent stem cells (hPSCs) underchemically defined, albumin-free conditions. Activation of Wnt signalinginduces mesodermal differentiation, while subsequent Wnt inhibitiondrives cardiac specification. Reactivation of Wnt in cardiac progenitorsspecifies epicardial cells. These hPSC-derived epicardial cells retainmany characteristics of primary epicardial cells, including formation ofa polarized epithelial sheet, expression of key epicardial genes Wilms'tumor suppressor protein 1 (WT1), TCF21, TBX18, and ALDH1A2, and theability to undergo epithelial-to-mesenchymal transition (EMT) togenerate fibroblast and vascular smooth muscle lineages. In addition,inhibition of TGFβ pathway signaling via culture in the presence ofsmall molecules permits long-term maintenance of hPSC-derived epicardialcells in vitro. The chemically defined platform described here should bewidely useful for generating functional epicardial cells for bothresearch and clinical applications. The epicardial differentiationprocess provided herein is chemically defined and albumin-free, andgenerates epicardial cells from human pluripotent stem cells at agreater yield and lower cost than existing methods. In addition, thesecells are more suitable for translational applications since they arederived in the absence of xenogeneic components. The methods providedherein have valuable applications such as inexpensive and reproduciblegeneration of human epicardial cells. Generating human epicardial cellsin completely chemically-defined, xeno-free conditions can facilitatetranslation of these cells to regenerative therapies and other clinicalapplications. As described in further detail below, the Inventors'xenogeneic material-free, albumin-free protocols target key regulatoryelements of the Wnt/β-catenin signaling pathway, simplifying the stepsand components involved in deriving cardiomyocyte progenitors andepicardial cells from pluripotent stem cells.

Accordingly, in a first aspect, provided herein is a method forgenerating a population of human epicardial cells, where the methodcomprises differentiating cardiac progenitor cells (such as humanpluripotent stem cell-derived cardiac progenitor cells) under conditionsthat promote differentiation of thecardiac progenitors into epicardialcells. As used herein, the term “epicardial cells” refers to cells ofthe epicardial lineage obtained according to a method provided herein.Epicardial cells are characterized and identified by expression oftranscription factors including Wilms' tumor suppressor protein (WT1),TCF21, and TBX18. WT1 is a transcription factor expressed in epicardiumand epicardium-derived cells (EPDC) as well as in many cells of theearly proepicardium (PE).

Preferably, the method comprises (i) generating or obtaining apopulation of cardiac progenitor cells; (ii) activating Wnt/β-cateninsignaling in the cardiac progenitor cells of step (i) for a period ofabout two days; and (iii) culturing the population of step (ii) forabout two days to about ten days to obtain a cell population comprisingepicardial cells. Any human cardiac progenitor cell can be usedaccording to the methods provided herein as long as the cardiacprogenitor cell is positive for the expression of one or more of thefollowing cardiac lineage markers: Isl1, Flk-1, and Nkx2.5. In somecases, the cardiac progenitor cells for step (ii) are passaged orunpassaged cardiac progenitors cells, where the passaged cells are fromculture day 4, day 5, day 6, or day 7. As described herein, exogenousTGFβ superfamily growth factors such as Bone Morphogenetic Protein 4(BMP4) are not required to generate cells of the epicardial lineage fromhuman pluripotent cells.

As will be appreciated by those of ordinary skill in the art,Wnt/β-catenin signaling can be activated by modulating the function ofone or more proteins that participate in the Wnt/β-catenin signalingpathway to increase β-catenin expression levels or activity, TCF and LEFexpression levels, or β-catenin/TCF/LEF induced transcriptionalactivity.

In some embodiments, activation of Wnt/β-catenin signaling is achievedby inhibiting Gsk3 phosphotransferase activity or Gsk3 bindinginteractions. While not wishing to be bound by theory, it is believedthat inhibition of Gsk3 phosphorylation of β-catenin will inhibit tonicdegradation of β-catenin and thereby increase the level of β-catenin andactivity to drive differentiation of pluripotent stem cells to anendodermal/mesodermal lineage. Gsk3 inhibition can be achieved in avariety of ways including, but not limited to, providing small moleculesthat inhibit Gsk3 phosphotransferase activity, RNA interferenceknockdown of Gsk3, and overexpression of dominant negative form of Gsk3.Dominant negative forms of Gsk3 are known in the art as described, e.g.,in Hagen et al. (2002), J Biol Chem, 277(26):23330-23335, whichdescribes a Gsk3 comprising a R96A mutation.

In some embodiments, Gsk3 is inhibited by contacting a cell with a smallmolecule that inhibits Gsk3 phosphotransferase activity or Gsk3 bindinginteractions. Suitable small molecule Gsk3 inhibitors include, but arenot limited to, CHIR99021, CHIR98014, BIO-acetoxime, BIO, LiCl, SB216763, SB 415286, AR A014418, 1-Azakenpaullone, Bis-7-indolylmaleimide,and any combinations thereof. In some embodiments, any of CHIR99021,CHIR98014, and BIO-acetoxime are used to inhibit Gsk3 in pluripotentstem cells in the differentiation methods described herein. In oneembodiment, the small molecule Gsk3 inhibitor to be used is CHIR99021 ata concentration ranging from about 3 μM to about 9 μM, e.g., about 3 μM,4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM or another concentration of CHIR99021from about 3 μM to about 9 μM. In another embodiment, the small moleculeGsk3 inhibitor to be used is CHIR98014 at a concentration ranging fromabout 0.1 μM to about 1 μM, e.g., about 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM,0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM or another concentration ofCHIR98014 from about 0.1 μM to about 1 μM. In another embodiment, thesmall molecule Gsk3 inhibitor to be used is BIO-acetoxime at aconcentration ranging from about 0.1 μM to about 1 μM, e.g., about 0.1μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM oranother concentration of BIO-acetoxime from about 0.1 μM to about 1 μM.

In other embodiments, Gsk3 activity is inhibited by RNA interferenceknockdown of Gsk3. For example, Gsk3 expression levels can beknocked-down using commercially available siRNAs against Gsk3, e.g.,SignalSilence® GSK-3α/β siRNA (catalog #6301 from Cell SignalingTechnology®, Danvers, Mass.), or a retroviral vector with an inducibleexpression cassette for Gsk3, e.g., a commercially availableTet-inducible retroviral RNA interference (RNAi) system from Clontech(Mountain View, Calif.) Catalog No. 630926, or a cumate-inducible systemfrom Systems Biosciences, Inc. (Mountain View, Calif.), e.g., the SparQ®system, catalog no. QM200PA-2.

In other embodiments, the Wnt/β-catenin signaling pathway is activatedby overexpressing β-catenin itself, e.g., human β-catenin (exemplarynucleotide and amino acid sequences are found at GenBank Accession Nos:X87838 and CAA61107.1, respectively). In one embodiment, β-cateninoverexpression is achieved using an inducible expression system, e.g.,any of the just-mentioned inducible expression systems. Alternatively, aconstitutively active, stabilized isoform of β-catenin is used, whichcontains point mutations S33A, S37A, T41A, and S45A as described, e.g.,in Baba et al. (2005), Immunity 23(6):599-609.

In yet other embodiments, Wnt/β-catenin signaling pathway activation inpluripotent stem cells is achieved by contacting the cells with an agentthat disrupts the interaction of β-catenin with Axin, a member of theβ-catenin destruction complex. Disruption of the Axin/β-catenininteraction allows β-catenin to escape degradation by the destructioncomplex thereby increasing the net level of β-catenin to drive β-cateninsignaling. For example, the Axin/β-catenin interaction can be disruptedin pluripotent cells by contacting the cells with the compound5-(Furan-2-yl)-N-(3-(1H-imidazol-1-yl)propyl)-1,2-oxazole-3-carboxamide(“SKL2001”), which is commercially available, e.g., as catalog no.681667 from EMD4 Biosciences. An effective concentration of SKL2001 toactivate Wnt/β-catenin signaling ranges from about 10 μM to about 100μM, about 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM oranother concentration of SKL2001 from about 10 μM to about 100 μM.

In some embodiments, activation (or re-activation) of Wnt/β-cateninpathway is initiated 36 hours following the inhibition step e.g., atleast about 36 hours to about 72 hours after the beginning of theinhibition step.

The methods provided herein produce isolated populations of pluripotentstem cell-derived epicardial cells, where the isolated population is asubstantially pure population of epicardial cells. As used herein,“isolating” and “isolated” refer to separating, selecting, or enrichingfor a cell type of interest or subpopulation of cells from surrounding,neighboring, or contaminating cells or from cells of another type. Asused herein, the term “substantially pure” refers to a population ofcells that is at least about 75% (e.g., at least about 75%, 85%, 90%,95%, 98%, 99% or more) pure, with respect to epicardial cells making upa total cell population. In other words, the term “substantially pure”refers to a population of epicardial cells of the present invention thatcontains fewer than about 20%, fewer than about 10%, or fewer than about5% of non-epicardial cells (e.g., cardiomyocytes) when directingdifferentiation to obtain cells of the epicardial lineage. The term“substantially pure” also refers to a population of epicardial cells ofthe present invention that contains fewer than about 20%, about 10%, orabout 5% of non-epicardial cells in an isolated population prior to anyenrichment, expansion step, or differentiation step. Typically, apopulation comprising epicardial cells obtained by the disclosed methodscomprises a very high proportion of epicardial cells. In someembodiments, the cell population comprises about 50% to about 99%epicardial cells, e.g., about 52%, 55%, 67%, 70%, 72%, 75%, 80%, 85%,90%, 95%, 98%, or another percent of epicardial cells from about 50% toabout 99% epicardial cells.

Epicardial cells can be identified by the presence of one or moreepicardial markers. Useful gene expression or protein markers foridentifying epicardial cells include, but are not limited to, Wilms'tumor suppressor protein (WT1), TCF21, Tbx18, and combinations thereof.Preferably, the method yields a cell population, at least 95% (e.g., atleast 95%, 96%, 97%, 98%, 99% or more) of which are epicardial cellspositive for expression of Wilms' tumor suppressor protein (WT1).Molecular markers of the epicardium can be detected at the mRNAexpression level or protein level by standard methods in the art. Insome embodiments, no cell separation step or method is used to obtain asecond cell population comprising at least 70% WT1⁺ cells or at least85% WT1⁺ cells. In other embodiments, the proportion of epicardial cellsin a population of cells obtained in the described methods is enrichedusing a cell separation, cell sorting, or enrichment method, e.g.,fluorescence activated cell sorting (FACS), enzyme-linked immunosorbentassay (ELISA), magnetic beads, magnetic activated cell sorting (MACS),laser-targeted ablation of non-epicardial cells, and combinationsthereof. Preferably, FACS is used to identify and separate cells basedon cell-surface antigen expression. In some embodiments, certainepicardial functional criteria are also assessed. Such functionalepicardial cell criteria include, without limitation, formation of apolarized epithelial sheet and the ability to undergoepithelial-to-mesenchymal transition (EMT) (in vitro or in vivo) togenerate fibroblast and vascular smooth muscle lineages.

As described above, any human cardiac progenitor cell can be usedaccording to the methods provided herein as long as the cardiacprogenitor cell is positive for the expression of one or more of thefollowing cardiac lineage markers: Isl1, Flk-1, and Nkx2.5. In exemplaryembodiments, human cardiac progenitor cells are obtained by directingdifferentiation of human pluripotent stem cells into the mesodermal andcardiac lineages. In such cases, the method comprises the steps of: (a)activating Wnt/β-catenin signaling in human pluripotent stem cells byculturing the human pluripotent stem cells to obtain a first cellpopulation; and (b) inhibiting Wnt/β-catenin signaling in the first cellpopulation by culturing the first cell population in the presence of anagent that inhibits canonical Wnt/β-catenin pathway signaling to obtaina second cell population comprising human cardiac progenitor cells.Preferably, each culturing step is performed under chemically defined,xeno-free, and albumin-free conditions. For purposes of this disclosure,“xeno-free” means having no xenogeneic products of non-human animalorigin, such as cells, tissues and/or body fluids, or any tissue orblood components, such as serum, which contain variable and undefinedfactors. Xeno-free medium and culture substrates are made up of known or“defined” components, which reduces the risk of viral contamination,prion transmission, and the batch-to-batch variability that is presentusing an undefined medium. Accordingly, for human cells, a xeno-freeculture medium is defined as a culture medium essentially free of animalcomponents, wherein the animal is not a human.

In exemplary embodiments, human pluripotent stem cell-derived cardiacprogenitor cells are singularized and replated at a low cell density ina basal culture medium. Optionally, such single cell replating isfollowed by period of outgrowth before proceeding to activation ofWnt/β-catenin pathway signaling according to a method of generatingepicardial cells as provided herein.

As will be appreciated by those of ordinary skill in the art,Wnt/β-catenin signaling can be inhibiting by modulating the function ofone or more proteins that participate in the Wnt/β-catenin signalingpathway to decrease β-catenin levels or activity, decrease TCF and LEFexpression levels, or decrease β-catenin/TCF-LEF-induced transcriptionalactivity. For example, inhibition of Wnt/β-catenin pathway signalingincludes inhibition of TCF/LEF-β-catenin mediated gene transcription.Wnt/β-catenin pathway signaling can be inhibited in various waysincluding but not limited to providing small molecule inhibitors, RNAinterference, or blocking antibodies against functional canonical Wntligands or Wnt pathway receptors (e.g., Frizzled and LRP5/6); providingsmall molecules that promote degradation of β-catenin and/or TCF/LEF;gene interference knockdown of β-catenin and/or TCF/LEF; overexpressionof a dominant negative form of β-catenin lacking the sequence forbinding to TCF/LEF; overexpressing Axin2 (which increases β-catenindegradation); providing a small molecule inhibitor of a TCF/LEF andβ-catenin interaction; and providing a small molecule inhibitor of aTCF/LEF-β-catenin and DNA promoter sequence interaction.

In some cases, Wnt/β-catenin pathway signaling is inhibited bycontacting a cell with one or more small molecule inhibitors of a Wntligand (e.g., a small molecule that inhibits secretion of the Wntligand) by inhibiting interactions between a Wnt ligand and itsreceptor. For example, the small molecule that inhibits Wnt/β cateninsignaling can be a small molecule that prevents palmitoylation of Wntproteins by porcupine (i.e., a porcupine inhibitor). In someembodiments, the small molecule that prevents palmitoylation of Wntproteins by porcupine includesN-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide(“IWP2”),2-(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidin-2-ylthio)-N-(6-methylbenzo[d]thiazol-2-yl)acetamide(“IWP4”),4-(2-Methyl-4-pyridinyl)-N-[4-(3-pyridinyl)phenyl]benzeneacetamide(“Wnt-C59”), or a combination thereof. In some such embodiments, theporcupine inhibitor is present in a concentration of from 0.2 μM to 5μM. Other Wnt signaling inhibitors are available commercially, e.g., asSigma catalog no. I0161; Benzoic acid, 2-phenoxy-,2-[(5-methyl-2-furanyl)methylene]hydrazide (“PNU-74654”), e.g., Sigmacatalog no. P0052.

In exemplary embodiments, a human pluripotent stem cell-derivedmesodermal cell obtained according to step (a) as set forth above (e.g.,cells characterized by the expression of mesodermal molecular markerBrachyury/T) is cultured in the presence of an agent that inhibitsWnt/β-catenin pathway signaling for about 8 hours to about 48 hours,e.g., about 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 28 hours,32 hours, 36 hours, 40 hours, 44 hours, 48 hours, or another period ofWnt/β-catenin pathway signaling inhibition from about 8 hours to about48 hours to obtain a population of cells. In one embodiment, the humanpluripotent stem cell-derived cells characterized by the expression ofmesodermal molecular markers are subjected to Wnt/β-catenin pathwaysignaling inhibition for about 24 hours.

In other cases, a human pluripotent stem cell-derived mesodermal cellobtained according to step (a) as set forth above (e.g., cellscharacterized by the expression of mesodermal molecular markersBrachyury/T is cultured in the presence of one or more small moleculecompounds that promote degradation of β-catenin. In some cases, suchsmall molecule compounds are compounds that, directly or indirectly,stabilize Axin, which is a member of the β-catenin destruction complex,and thereby enhance degradation of β-catenin. Examples ofAxin-stabilizing compounds include but are not limited to3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one(“XAV939”), e.g., Sigma catalog no. X3004;4-(1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-Benzamide(“IWR-1”) available commercially, e.g., as Sigma catalog no. I0161. Insome cases, such small molecule compounds are compounds that, directlyor indirectly, activate casein kinase 1α (CK1), which is a member of theβ-catenin destruction complex, and thereby enhance degradation ofβ-catenin. Examples of CK1-stabilizing compounds include but are notlimited to6-(Dimethylamino)-2-[2-(2,5-dimethyl-1-phenyl-1H-pyrrol-3-yl)ethenyl]-1-methyl-4,4′-methylenebis[3-hydroxy-2-naphthalenecarboxylate](2:1)-quinolinium (“Pyrvinium pamoate salt hydrate”), e.g., Sigmacatalog no. P0027.

Typically, inhibition of Wnt/β-catenin signaling during step (ii) ismaintained for at least about 1 day to about 6 days, e.g., about 1 day,2 days, 2.5 days, 3 days, 3.5 days, 4 days, 5 days, or another period ofWnt/β-catenin signaling inhibition from at least about 1.5 days to about6 days. In some embodiments, where a small molecule is used to inhibitWnt/β-catenin signaling, such cells are contacted with a small moleculeinhibitor of Wnt/β-catenin signaling for about 2 days, and then cultureof this cell population continues in the substantial absence of thesmall molecule inhibitor. In other embodiments, where inducible RNAinterference is used (e.g., with an inducing agent such as doxycyclineto drive expression of tet-on expression cassette) to knockdownexpression of β-catenin, induction and maintenance of β-catenin shRNAexpression occurs for about 3.5 days, after which induction of β-cateninshRNA expression is terminated, and then culture of the first cellpopulation continues in the substantial absence of the shRNA inducingagent.

A suitable working concentration range for small molecule Wnt/β-cateninsignaling inhibitors is from about 0.1 μM to about 100 μM, e.g., about 2μM, 5 μM, 7 μM, 10 μM, 12 μM, 15 μM, 18 μM, or another workingconcentration of one or more of the foregoing small molecule inhibitorsranging from about 0.1 μM to about 100 μM. For example, IWP2 or IWP4 orWnt-C59 can be used at a working concentration of from about 1 to 4 μM.In another embodiment, IWP2 or IWP4 or Wnt-C59 is used at a workingconcentration of about 2.5 μM. In other embodiments, one or more of theabove-mentioned small molecule inhibitors is used at the correspondingtarget IC₅₀.

In other embodiments, inhibition of Wnt/β-catenin pathway signaling isachieved using RNA interference to decrease the expression of one ormore targets in the Wnt/β-catenin pathway. For example, in some cases,RNA interference is against β-catenin itself. In one embodiment, whereone or more short hairpin interfering RNAs (shRNAs) knock down β-cateninexpression, at least one of the following shRNA sequences is used:5′-CCGGAGGTGCTATCTGTCTGCTCTACTCGAGTAGAGCAGACAGATAGCACCTTTTTT-3′ (SEQ IDNO:1) or 5′-CCGGGCTTGGAATGAGACTGCTGATCTCGAGATCAGCAGTCTCATTCCAAGCTTTTT-3′(SEQ ID NO:2). Such shRNAs may be transfected as synthetic shRNAs intothe first cell population by a number of standard methods known in theart. Alternatively, shRNA sequences may be expressed from an expressionvector, e.g., from a plasmid expression vector, a recombinantretrovirus, or a recombinant lentivirus.

In some cases, an inducible expression cassette is used to express aninterfering RNA, e.g., an shRNA against β-catenin, as exemplifiedherein. The use of an inducible expression cassette allows temporalcontrol of β-catenin knockdown. Such temporal control is well suited tothe timing of Wnt/β-catenin signaling inhibition used in thedifferentiation methods described herein.

In an alternative method, Wnt/β-catenin signaling is inhibited using atleast one antibody that blocks activation of a Wnt ligand receptor orbinds to one or more Wnt ligand family members. Such antibodies areknown in the art, as described in, e.g., an anti-Wnt-1 antibodydescribed in He et al. (2004), Neoplasia 6(1):7-14. In otherembodiments, the blocking antibody is targeted against a Wnt ligandreceptor and blocks the interaction of Wnt ligands with the receptor, asdescribed, e.g., in Gurney et al. (2012), Proc. Natl. Acad. Sci. USA,109(29): 11717-22.

As used herein, “pluripotent stem cells” appropriate for use accordingto a method of the invention are cells having the capacity todifferentiate into cells of all three germ layers. Pluripotent stemcells (PSCs) suitable for the differentiation methods disclosed hereininclude, but are not limited to, human embryonic stem cells (hESCs),human induced pluripotent stem cells (hiPSCs), non-human primateembryonic stem cells (nhpESCs), non-human primate induced pluripotentstem cells (nhpiPSCs). As used herein, “embryonic stem cells” or “ESCs”mean a pluripotent cell or population of pluripotent cells derived froman inner cell mass of a blastocyst. See Thomson et al., Science282:1145-1147 (1998). These cells express Oct-4, SSEA-3, SSEA-4,TRA-1-60, and TRA-1-81, and appear as compact colonies having a highnucleus to cytoplasm ratio and prominent nucleolus. ESCs arecommercially available from sources such as WiCell Research Institute(Madison, Wis.).

As used herein, “induced pluripotent stem cells” or “iPS cells” mean apluripotent cell or population of pluripotent cells that may vary withrespect to their differentiated somatic cell of origin, that may varywith respect to a specific set of potency-determining factors and thatmay vary with respect to culture conditions used to isolate them, butnonetheless are substantially genetically identical to their respectivedifferentiated somatic cell of origin and display characteristicssimilar to higher potency cells, such as ESCs, as described herein. See,e.g., Yu et al., Science 318:1917-1920 (2007). Induced pluripotent stemcells exhibit morphological properties (e.g., round shape, largenucleoli and scant cytoplasm) and growth properties (e.g., doubling timeof about seventeen to eighteen hours) akin to ESCs. In addition, iPScells express pluripotent cell-specific markers (e.g., Oct-4, SSEA-3,SSEA-4, Tra-1-60, or Tra-1-81, but not SSEA-1). Induced pluripotent stemcells, however, are not immediately derived from embryos. As usedherein, “not immediately derived from embryos” means that the startingcell type for producing iPS cells is a non-embryonic, non-pluripotentcell, such as a multipotent cell or terminally differentiated cell, suchas somatic cells obtained from a post-natal individual.

Human iPS cells can be used according to a method described herein toobtain epicardial cells having the genetic complement of a particularhuman subject. For example, it may be advantageous to obtain epicardialcells that exhibit one or more specific phenotypes associated with orresulting from a particular disease or disorder of the particularmammalian subject. In such cases, iPS cells are obtained byreprogramming a somatic cell of a particular human subject according tomethods known in the art. See, for example, Yu et al., Science324(5928):797-801 (2009); Chen et al., Nat. Methods 8(5):424-9 (2011);Ebert et al., Nature 457(7227):277-80 (2009); Howden et al., Proc. Natl.Acad. Sci. U.S.A. 108(16):6537-42 (2011). Induced pluripotent stemcell-derived epicardial cells allow modeling of drug responses inepicardial cells obtained from an individual having, for example, aparticular disease. Even the safest drugs may cause adverse reactions incertain individuals with a specific genetic background or environmentalhistory. Accordingly, human subject specific iPS cell-derived epicardialcells are useful to identify genetic factors and epigenetic influencesthat contribute to variable drug responses.

Subject-specific somatic cells for reprogramming into iPS cells can beobtained or isolated from a target tissue of interest by biopsy or othertissue sampling methods. In some cases, subject-specific cells aremanipulated in vitro prior to use. For example, subject-specific cellscan be expanded, differentiated, genetically modified, contacted topolypeptides, nucleic acids, or other factors, cryo-preserved, orotherwise modified.

Chemically defined culture medium and substrate conditions for culturingpluripotent stem cells, as used in the methods described herein, arewell known in the art. Preferably, a serum-free, chemically defined,albumin-free culture medium is used. As used herein, the terms“chemically-defined culture conditions,” “fully defined, growth factorfree culture conditions,” and “fully-defined conditions” indicate thatthe identity and quantity of each medium ingredient is known and theidentity and quantity of supportive surface is known. As used herein,“serum-free” means that a medium does not contain serum or serumreplacement, or that it contains essentially no serum or serumreplacement. For example, an essentially serum-free medium can containless than about 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or0.1% serum. As used herein, the term “albumin-free conditions” indicatesthat the culture medium used contains no added albumin in any form,including without limitation Bovine Serum Albumin (BSA) or any form ofrecombinant albumin.

In some embodiments, pluripotent stem cells to be differentiatedaccording to the methods disclosed herein are cultured in the presenceof a serum-free, albumin-free, chemically-defined culture medium such asLaSR basal medium (a serum-free culture medium containing AdvancedDMEM/F12, 2.5 mM GlutaMAX, and supplemented with 60 μg/mL ascorbicacid), mTESR-1® medium (StemCell Technologies, Inc., Vancouver, Calif.),or Essential 8® medium (Life Technologies, Inc.) on a Matrigel™substrate (BD Biosciences, NJ) or Synthemax surfaces (Corning) accordingto the manufacturer's protocol. A number of known basal culture mediaare suitable for use throughout the differentiation methods describedherein. Such basal cell culture media include, but are not limited to,RPMI, DMEM/F12 (1:3), DMEM/F12 (1:1), DMEM/F12 (3:1), F12, DMEM, andMEM. In exemplary embodiments, these basal cell culture media aresupplemented with 50 to 200 μg/ml L-Ascorbic acid 2-phosphatesesquimagnesium salt hydrate (e.g., Sigma, catalog no. A8960). For eachdifferentiation step described herein, cells are cultured in a mediumthat is substantially free of exogenous Bone Morphogenetic Proteins(BMPs) such as BMP4.

In exemplary embodiments, human pluripotent stem cells (e.g., human ESCsor iPS cells) are cultured in the absence of a feeder layer (e.g., afibroblast layer) and in the presence of a chemically defined,xenogen-free (“xeno-free”) substrate. For example, human pluripotentcells can be cultured in the presence of a substrate comprisingvitronectin, a vitronectin fragment or variant, a vitronectin peptide, aself-coating substrate such as Synthemax® (Corning), or combinationsthereof. In exemplary embodiments, the chemically-defined, xeno-freesubstrate is a plate coated in vitronectin peptides or polypeptides(e.g., recombinant human vitronectin).

In a further aspect, provided herein are compositions and methods forexpanding a self-renewing population of human epicardial cells for anextensive period of time. This is because the invention providescompositions and methods of generating human epicardial cells whichcanbe extensively expanded in vitro yet retain the ability to undergo anepithelial-to-mesenchymal transition (EMT) and differentiate intoepicardium-derived cell types. The term “extensively expanded” as usedherein refers to cell populations which have undergone at least about 25or more cell population doublings and wherein the cells arenon-senescent and are not immortalized. When cultured in the presence ofan expansion medium comprising an inhibitor of TGFβ, epicardial cellsobtained according to the methods provided herein are capable ofundergoing at least 25 cell divisions (e.g., at least 25, 30, 35, ormore cell divisions). Inhibitors of TGFB signaling that can be usedinclude, without limitation, A83-01 or SB431542. As described in theExamples that follow, culture in the presence of an expansion mediumcomprising of A83-01 or SB431542 yielded hPSC-derived epicardial cellscapable of at least 25 population doublings, generating more than 10million cells from a single hPSC-derived epicardial cell clone. In somecases, a cell culture comprises a chemically defined, albumin-freeexpansion medium comprising an inhibitor of TGFβ signaling, and humanself-renewing epicardial cells that proliferate in culture and maintainthe ability to undergo EMT, where the epicardial cells are notimmortalized. Therefore, provided herein is an expandable source offunctional epicardial cells.

Articles of Manufacture

In another aspect, provided herein is a kit for generating humanepicardial cells. In exemplary embodiments, the kit comprises (i) aculture medium suitable for differentiating human cardiac progenitorcells into epicardial cells; (ii) an agent that activates Wnt signalingin human cardiac progenitor cells; and (iii) instructions describing amethod for generating human epicardial cells, the method employing theculture medium and the agent. In some cases, a kit provided hereinfurther comprises or alternatively comprises instructions describingmethods for long-term in vitro maintenance of human epicardial cellsobtained according to a kit provided herein, where the method employs aculture medium suitable for maintaining human epicardial cells and anagent that supports long-term maintenance of such epicardial cells.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar to or equivalent to those described herein can be usedin the practice or testing of the present invention, preferred methodsand materials are described herein.

In describing the embodiments and claiming the invention, the followingterminology will be used in accordance with the definitions set outbelow.

As used herein, “about” means within 5% of a stated concentration rangeor within 5% of a stated time frame.

As used herein, “a medium consisting essentially of” means a medium thatcontains the specified ingredients and those that do not materiallyaffect its basic characteristics.

The terms “defined culture medium,” “defined medium,” and the like, asused herein, indicate that the identity and quantity of each mediumingredient is known. The term “defined,” when used in relation to aculture medium or a culture condition, refers to a culture medium or aculture condition in which the nature and amounts of approximately allthe components are known.

As used herein, “effective amount” means an amount of an agentsufficient to evoke a specified cellular effect according to the presentinvention.

Cells are “substantially free” of exogenous genetic elements or vectorelements, as used herein, when they have less that 10% of theelement(s), and are “essentially free” of exogenous genetic elements orvector elements when they have less than 1% of the element(s). However,even more desirable are cell populations wherein less than 0.5% or lessthan 0.1% of the total cell population comprise exogenous geneticelements or vector elements. A culture, composition, or culture mediumis “essentially free” of certain reagents, such as signaling inhibitors,animal components or feeder cells, when the culture, composition, andmedium, respectively, have a level of these reagents lower than adetectable level using conventional detection methods known to a personof ordinary skill in the art or these agents have not been extrinsicallyadded to the culture, composition, or medium.

The invention will be more fully understood upon consideration of thefollowing non-limiting Examples. It is specifically contemplated thatthe methods disclosed are suited for pluripotent stem cells generally.All publications, patents, and patent applications disclosed herein arehereby incorporated by reference as if set forth in their entirety.

EXAMPLES Example 1: Epicardial Lineage-Specific Differentiation of HumanPluripotent Stem Cells

In this Example, we demonstrate that Wnt signaling and inhibition ofTGFβ signaling is sufficient for epicardial induction and self-renewal,respectively, from hPSCs under chemically-defined, animal component-freeconditions. The hPSC-derived epicardial cells were similar to primaryepicardial cells both in vitro and in vivo. These findings improve ourunderstanding of self-renewal mechanisms of the epicardium and haveimplications for stimulating epicardial regeneration via cellular orsmall molecule therapies. These hPSC-derived epicardial cells retainmany characteristics of primary epicardial cells, including formation ofa polarized epithelial sheet, expression of key epicardial genes such asWilms' tumor suppressor protein (WT1), TBX18, and ALDH1A2, and theability to undergo epithelial-to-mesenchymal transition (EMT) both invitro and in vivo to generate fibroblast and vascular smooth musclelineages. The chemically-defined platform described here rapidlygenerates epicardial cells from human pluripotent stem cells (hPSCs) andenables the assessment of the temporal roles of different signalingpathways during epicardium formation and proliferation.

Methods

Construction of Donor Plasmid and sgRNA:

Human codon-optimized Streptococcus pyogenes wild-type Cas9(pCas9-2A-eGFP #) was obtained from Addgene (plasmid #44719) andchimeric guide RNA expression cassette was cloned into this Cas9-2A-eGFPplasmid with two BbsI restriction sites for rapid sgRNA cloning. TwosgRNAs targeting at or near WT1 stop codon (1: AACTCCAGCTGGCGCTTTGAGGG(SEQ ID NO:3) and 2: GGACACTGAACGGTCCCCGAGGG (SEQ ID NO:4)) were used.To generate the WT1-2A-eGFP donor plasmid, DNA fragments of about 2 kbin length were PCR amplified from the genomic DNA before and after thestop codon of WT1 and were cloned into the OCT4-2A-eGFP donor plasmid²⁹(Addgene #31938), replacing the OCT4 homologous arms.

Maintenance of hPSCs and TAT-Cre Treatment of WT1 Knock-in hPSCs:

Transgene and vector-free human pluripotent stem cells (hPSCs) weremaintained on Matrigel™ (Corning) or SyntheMax® (BD Biosciences)-coatedplates in mTeSR1 orE8 medium (STEMCELL Technologies) according topreviously published methods^(18,30). To remove PGK-Puro cassette fromthe WT1-2A-eGFP cells, targeted homozygous clones were treated with 2 μMTAT Cre Recombinase (Excellgen, EG-1001) for 6 hours in E8 medium. Aftertwo days, cells were singularized with Accutase and seeded intoMatrigel™-coated 96-well plate at a density of 100 to 150 cells perwell. After two weeks, cells were subjected for PCR genotyping.

Electroporation:

Pre-treated hESCs with 10 μM ROCK inhibitor (Y27632) for 3 to 4 hoursprior to electroporation. Cells were digested by Accutase (InnovativeCell Technologies) at 37° C. for 8 minutes and 2.5-3 million singlecells were electroporated with 3 μg gRNA1, 3 μg gRNA2 and 6 μgWT1-2A-eGFP donor plasmids in 200 μL cold PBS−/− using the Gene PulserXcell System (Bio-Rad) at 320 V, 200 μF and 1000′Ω (Time constant shouldbe around 15 ms) in a 0.4 cm cuvette. Two electroporation were performedand approximately 5-6 million cells were subsequently plated ontoMatrigel™-coated 10-cm dish in 10 mL mTeSR1 with 10 μM Y27632. 24 hourslater, and every day afterwards, the medium was changed with freshmTeSR1. Three days after electroporation, 1 μg/ml puromycin was addedinto the mTeSR1 for selection for about two weeks. Single cell cloneswere then picked into Matrigel™-coated 96-well plate and subjected forPCR genotyping after 4 to 7 days.

Cardiac Progenitor Induction Via Modulation of Canonical Wnt Signaling:

When hPSCs maintained on a SyntheMax-coated surface achieved confluence,cells were singularized with Accutase (Innovative Cell Technologies) at37° C. for 5 minutes and then seeded onto a SyntheMax-coated cellculture dish at 250,000 cells/cm² in mTeSR1 or E8 supplemented with 5 μMROCK inhibitor Y-27632 (Selleckchem) (day −3) for 24 hours. Cells werethen cultured in mTeSR1 or E8, changed daily. At day 0, cells weretreated with 6 μM CHIR99021 (Selleckchem) for 24 hours in RPMI medium,followed by a change with RPMI medium at day 1. 2.5-5 μM IWP2 (Tocris)was added at day 3 and removed during the medium change at day 5.

Epicardial Cell Generation Via Activation of Canonical Wnt Signaling:

At day 6, cardiac progenitor cells were singularized with Accutase at37° C. for 5 minutes and then seeded onto a gelatin-coated cell culturedish at 20,000-80,000 cells/cm² in LaSR basal medium (advanced DMEM/F12with 100 μg/mL ascorbic acid) or RPMI/Vc/Ins medium (100 μg/mL ascorbicacid and 1 μg/mL human recombinant or bovine insulin (Sigma)) with 5 μMROCK inhibitor Y-27632 for 24 hours. At day 7, cells were treated with1-9 μM CHIR99021 for 2 days in LaSR basal medium or RPMI/insulin/Vcmedium. After 2 days, CHIR99021-containing medium was aspirated andcells were cultured in LaSR basal medium or RPMI/insulin/Vc mediumwithout CHIR99021 for 3-5 additional days.

Long-Term Maintenance of hPSC-Derived Epicardial Cells:

To expand the epicardial cells, confluent cells on day 3 or 4 ofdifferentiation were split 1:3 at a density of 0.04 to 0.08 millioncells/cm² using Versene (Life Technologies) or Accutase and routinelypassaged onto gelatin-coated plates in LaSR basal medium or RPMI/Vc/Insmedium and 0.5 μM A83-01 (Stemgent) or 2 μM SB431542 (Stemgent) withmedium changed daily until the cells reached confluence. Overnighttreatment of 5 μM Y27632 and 1% human recombinant albumin(Sigma-Aldrich) on single cells during passage was used to improve cellattachment and survival, but they were not required once cells attached.

Single Cell Passage and EMT Induction:

Confluent WT1+ cells were dissociated into single cells with Accutase at37° C. for 5 minutes and then seeded onto a gelatin-coated cell culturedish at 10,000 cells/cm² in LaSR basal medium supplemented with 5 μMROCK inhibitor Y-27632 for 24 hr. After 24 hours, medium was changed toLaSR basal medium and cells were treated with TGFβ1 or bFGF (R&DSystems) as indicated. Medium was changed every 3 days until analysis.

CF-ECM Scaffold to Transfer Epicardial Cells In Vivo to the InfarctedHeart:

Immunodeficient mice were purchased from Harlan Laboratories and allprocedures were carried out in accordance with protocols approved by theInstitutional Animal Care and Use Committee. Myocardial infarction wasinduced 48 hours before the transplantation according to a previouslydescribed protocol³¹. CF-ECM scaffolds were seeded with 1 million ES03eGFP hPSC-derived epicardial cells and incubated for 3 hours prior totransfer to the epicardial surface of the MI area. Aftertransplantation, the chest was closed. After 2, 6, 12 days, the mousehearts were harvested and excised for histology as previouslydescribed³¹.

Immunostaining Analysis:

Cells were fixed with 4% paraformaldehyde for 15 minutes at roomtemperature and then stained with primary and secondary antibodies(Table 1) in PBS plus 0.4% Triton X-100 and 5% non-fat dry milk(Bio-Rad). Nuclei were stained with Gold Anti-fade Reagent with DAPI(Invitrogen). An epifluorescence microscope (Leica DM IRB) with aQImaging® Retiga 4000R camera was used for imaging analysis.

Flow Cytometry Analysis:

Cells were dissociated into single cells with Accutase for 10 minutesand then fixed with 1% paraformaldehyde for 20 minutes at roomtemperature and stained with primary and secondary antibodies (Table 1)in PBS plus 0.1% Triton X-100 and 0.5% BSA. Data were collected on aFACSCaliber flow cytometer (Beckton Dickinson) and analyzed usingFlowJo. FACS gating was based on the corresponding isotype antibodycontrol.

Genomic DNA Extraction and Genomic PCR:

QuickExtract™ DNA Extraction Solution (Epicentre Cat. #QE09050) was usedto rapidly extract genomic DNA from hESCs according to manufactureinstructions. Genomic PCR was carried out using GoTaq Green Master Mix(Promega Cat. #M7123). PCR primer sequences are provided in the Table 2.

RT-PCR and Quantitative RT-PCR:

Total RNA was prepared with the RNeasy mini kit (QIAGEN) and treatedwith DNase (QIAGEN). 1 μg RNA was reverse transcribed into cDNA viaOligo (dT) with Superscript III Reverse Transcriptase (Invitrogen).Real-time quantitative PCR was done in triplicate with iQSYBR GreenSuperMix (Bio-Rad). GAPDH was used as an endogenous housekeepingcontrol. PCR primer sequences are provided in the Table 2.

RNA Sequencing and Data Analysis:

Total RNA of hPSC-derived epicardial was prepared with the Direct-zol™RNA MiniPrep Plus kit (Zymo Research) according to the manufactureinstructions. Human primary epicardial RNAs from 4 different donors wereprovided by our collaborator (AstraZeneca, Sweden). Samples wereperformed in IIIumina HiSeq2500 by Biotechnology Center at University ofWisconsin-Madison. The resulting sequence reads were mapped to the humangenome (hg19) using HISAT³², and the RefSeq transcript levels (RPKMs)were quantified using the python script rpmkforgenes.py³³. Hierarchicalclustering of whole transcripts were then plotted using GENE-E. Fastqfiles of hPSCs³⁴⁻³⁶, hPSC-derived ectoderm³⁵, endoderm³⁴, mesoderm³⁶ andCMs³⁷ were downloaded from Gene Expression Omnibus (GEO) (available atncbi.nlm.nih.gov/geo/on the World Wide Web) or ArrayExpress (availableat ebi.ac.uk/arrayexpress on the World Wide Web). Principal componentanalysis (PCA) was performed using PLS Toolbox 8.1 (EigenvectorTechnologies). The whole transcripts were preprocessed usingauto-scaling method (subtracting the mean from the variables anddividing by the standard deviation) to study the variance. Pathwayenrichment analysis was performed using Gene set enrichment analysis(GSEA) software³⁸. The gene expression data for each cell type wascompared with hPSCs and the significantly enriched pathways (p<0.05)were considered for further analysis. MATLAB 2013a (Mathworks Inc.) andMicrosoft Excel (2013) were used to identify the unique and commonpathways in different cell types. The absolute value of normalizedenrichment score (NES) of the top 50 significantly enriched pathways foreach cell type (ranked by the absolute NES) were further used forhierarchical clustering using GENE-E. To further investigate thesimilarity and differences in the number of enriched pathways in thethree cell types: donor derived epicardial cells, hPSC derivedepicardial cells and cardiomyocytes, top 150 significantly enrichedpathways for each cell type were selected.

Western Blot Analysis:

Cells were lysed in M-PER Mammalian Protein Extraction Reagent (Pierce)in the presence of Halt Protease and Phosphatase Inhibitor Cocktail(Pierce). Proteins were then separated by 10% Tris-Glycine SDS/PAGE(Invitrogen) under denaturing conditions and transferred to anitrocellulose membrane. After blocking with 5% non-fat milk in TBST,the membrane was incubated with primary antibody (Table 1) overnight at4° C. The membrane was then washed, incubated with an anti-mouse/rabbitperoxidase-conjugated secondary antibody for 1 hours at room temperatureor overnight at 4° C., and developed by SuperSignal chemiluminescence(Pierce).

Statistical Analysis:

Data are presented as mean±standard error of the mean (SEM). Statisticalsignificance was determined by Student's t-test (two-tail) between twogroups. P<0.05 was considered statistically significant.

Results

Chemically Defined Albumin-Free Conditions to GenerateISL1+NKX2.5+FLK-1+ Cardiac Progenitors

We previously demonstrated that temporal modulation of canonical Wntsignaling in RPMI basal medium (GiWi protocol) is sufficient to generatefunctional cardiomyocytes from hPSCs³⁹. We found that ISL1+NKX2.5+FLK-1+second heart field cardiac progenitor cells are generated asintermediates during the GiWi protocol (see FIG. 9A). When re-passagedon gelatin-coated plates in LaSR basal medium¹⁹ on day 6, thesehPSC-derived cardiac progenitors expressed progenitor markers includingISL1, NKX2.5 and FLK-1, as well as a proliferative marker KI67 (FIG.9B). Molecular analysis of cardiac progenitor differentiation from hPSCsrevealed dynamic changes in gene expression, with down-regulation of thepluripotency markers OCT4 and NANOG, and induction of the primitivestreak like gene T^(19,40) in the first 24 hr after CHIR99021 addition(FIG. 9C). Expression of cardiac progenitor markers ISL1, NKX2.5 andFLK-1 was first detected between days 3 and 5, and was significantlyup-regulated at day 6 (FIG. 9C).

Wnt/β-Catenin Signaling Regulates Specification of Epicardial Lineages

Pro-epicardium arises from ISL1+NKX2.5+ second heart field progenitorsin vivo^(1,41). To identify signaling mechanisms regulating cardiacprogenitor specification to epicardial cells, we treated day 6ISL1+NKX2.5+ progenitors with different small molecule and proteinmodulators of developmental signaling pathways for 48 hours (from day 7to day 9) (FIG. 1A, Table 3). hPSC-derived cardiac progenitors formedmore than 85% WT1+ putative epicardial cells following CHIR99021treatment (FIGS. 1B-C), demonstrating that Wnt signaling inductionbetween days 7 and 9 is sufficient to generate epicardial cells in theabsence of other exogenous signaling. In the absence of CHIR, robustbeating sheets of cTnT+ cardiomyocytes were observed (FIGS. 1B-C),suggesting that the activation status of canonical Wnt signaling at day7 toggles epicardial vs. cardiomyocyte differentiation. Interestingly,untreated and BMP4-, dorsomorphin (DM)- and retinoic acid (RA)-treatedcells also yielded about 10% WT1+ cells that were distinct from cTnT+cells; only 2% cTnT+WT1+ cells were generated (FIGS. 1B-C and FIG. 10).In the presence of CHIR, BMP4 treatment did not generate cardiomyocytes,but instead yielded an unknown population at the expense of WT1+ cells.Inhibition of BMP4 signaling via DM resulted in a similar purity of WT1+cells as CHIR treatment (FIG. 1B and FIG. 10), suggesting that BMP4signaling is dispensable at this stage of epicardial development.

Homozygous WT1-2A-eGFP Knock-in Reporter hPSCs Via CRISRP/Cas9Recapitulate the Epicardial Cell Differentiation Process

WT1 is required for the development of epicardium⁴² and the formation ofcardiovascular progenitor cellS⁴³. In order to better monitor theepicardial cell differentiation process and purify hPSC-derivedepicardial cells in vitro, we engineered the ES03 human embryonic stemcell line via CRISPR/Cas9-catalyzed homology-directed repair (HDR) andgenerated a homozygous WT1-2A-eGFP knock-in reporter cell line (FIG.2A). Two 2-kilobase homologous arm sequences located right before andafter WT1 stop codon were inserted into the Oct4-2A-eGFP donor plasmid⁴⁴and replaced the Oct4 homologous arms. We then introduced the 2A-eGFPsequence into the targeting sites by transfecting hESCs with theWT1-2A-eGFP donor plasmid and the Cas9/sgRNA plasmids. After puromycinselection, PCR genotyping and sequencing showed that ˜50% (21/44) of theclones were targeted in one (heterozygous) and ˜25% (12/44) both alleles(FIG. 2B). The homozygous clones were then subjected to TAT-Crerecombinase treatment and the PGK-Puro cassette was excised fromWT1-2A-eGFP (FIG. 2C). WT1-2A-eGFP-targeted hPSCs after Cre-mediatedexcision of the PGK-Puro cassette were subjected for CHIR treatment witheGFP detected at day 10 and boosted at day 12 (FIG. 2D). Dualimmunostaining of WT1 and GFP antibody resulted in expression of eGFP inWT1+ cells (FIG. 2E), demonstrating the success in generating WT1reporter cell line for potential cell tracking or purification.

Chemically Defined Albumin-Free Conditions to Generate WT1+ EpicardialCells

We next optimized the concentration of CHIR and initial seeding densityof cardiac progenitors at day 6 in LaSR basal medium, and found that 3μM CHIR with an initial density of 0.06 million cells per cm² yieldedmore than 95% WT1+ cells (FIGS. 3A-D), while 0 μM CHIR resulted in lessthan 10% WT1-eGFP. However, LaSR basal medium, which contains bovineserum albumin, adds xenogenic components to the medium which would notbe suitable for the generation of epicardial cells that meet clinicalrequirements. In order to develop a xeno-free protocol, wesystematically screened 4 commercially available basal mediasupplemented with 1 μg/mL human recombinant insulin and 100 μg/mLascorbic acid (Vc) as these two factors were shown to improve theculture of cardiac cell lineages⁴⁵⁻⁴⁷. DMEM, DMEM/F12 and RPMI generatedmore than 95% WT1+ putative epicardial cells from hPSC-derived cardiacprogenitors (FIG. 3E). To simplify the differentiation pipeline, weemployed RPMI as the basal medium, referring to epicardial cellgeneration from hPSCs as the “GiWiGi” (GSK3 inhibitor-WNT inhibitor-GSK3inhibitor) protocol.

Epicardial Cell Differentiation from Cardiac Progenitors is β-CateninDependent

Selectivity is a concern when using chemical inhibitors of signalingpathways. Therefore, we tested other GSK3 inhibitors includingBIO-acetoxime and CHIR98014 in the GiWiGi protocol, and found that 0.3μM CHIR98014 and BIO-acetoxime also induced WT1+ cell differentiation toa similar extent as 3 μM CHIR99021 (FIG. 4A). Although three smallmolecules, each with a distinct chemical structure, were used todecrease the likelihood of shared off-target effects, GSK3 inhibitionitself may affect other signaling pathways. In order to evaluate therole of β-catenin in GSK3 inhibitor-induced epicardial differentiation,we generated an iPSC cell line (19-9-11 ischcat-1) expressing β-cateninshRNA under the control of a tet-regulated inducible promoter. Upondoxycycline (dox) treatment, the shRNA efficiently down-regulatedβ-catenin expression (FIG. 4B). Our previous work¹⁵ showed that theinduction of NKX2.5+ISL1+ cardiac progenitors from hPSCs is β-catenindependent, therefore in this study we focused on the examination of thestage-specific roles of β-catenin during differentiation of epicardialcells from cardiac progenitors stimulated by GSK3 inhibition. We foundthat β-catenin knockdown at day 6 yielded significantly fewer WT1+cells, instead generating robust beating sheets of cTnT+ cardiomyocytesat the expense of WT1+ cells (FIG. 4C and FIG. 11). This finding isconsistent with reports that Wnt/β-catenin inhibition is necessary forcardiomyocyte formation from cardiac progenitors both in vitro and invivo^(15,25,48,49), and further supports the notion that Wnt/β-cateninsignaling regulates epicardial vs. cardiomyocyte specification. Theeffects of β-catenin knockdown on decreasing WT1+ cell generationgradually diminished after day 6, with no inhibition after day 9 (FIG.4C and FIG. 11).

Molecular Characterization of hPSC-Derived WT1+ Epicardial Cells

Pro-epicardial cells are marked by the expression of TBX18, WT1 andTCF21^(30,50,51). Molecular analysis of epicardial cell differentiationfrom hPSCs-derived cardiac progenitors (FIG. 5A and FIG. 12A) revealeddynamic changes in gene expression, with up-regulation of WT1 and TBX18,and undetectable TNNT2 (FIG. 5B). This was consistent with the WT1-eGFPsignals (FIG. 2D), and was also confirmed by western blot analysis ofWT1, TBX18 and TCF21 expression (FIG. 5C). Immunofluorescent analysisrevealed expression of pro-epicardial markers WT1, TBX18, and TCF21(FIGS. 5D-E). After passage at a low density, these cells adoptedcobblestone-like appearance typical of cultured primaryepicardium^(28,52) (FIG. 5E). In addition, the cells displayed intenseβ-catenin and ZO1 localization at sites of cell-to-cell contact. Takentogether, these data confirm the epithelial nature of these cells. Thesepost-passaged cells also expressed aldehyde dehydrogenase enzymeretinaldehyde dehydrogenase 2 (ALDH1A2) (FIGS. 12-A-C), suggesting theability to produce retinoic acids. Therefore, we refer to the day 12,pre-passaged WT1+ cells as pro-epicardial cells (Pro-Epi) and thepost-passaged WT1+ cells as epicardial cells (Epi). The GiWiGi protocolwas also effective in other hPSC lines, including human embryonic stemcell lines (hESC) H9 and ES03, and the 19-9-7 induced pluripotent stemcell (iPSC) line, generating more than 95% WT1+ cells (FIG. 13A).Post-passaged epicardial cells retained the expression of WT1 anddisplayed strong β-catenin and ZO1 staining along the cell borders (FIG.13B).

Long-Term Expansion of hPSC-Derived WT1+ Epicardial Cells

Primary mouse epicardial cells have been cultured for more than 3years⁵⁰, but epicardial cells isolated from the adult human heartrapidly undergo EMT in culture²⁸. Similar to primary human epicardialcells, hPSC-derived WT1+ epicardial cells only retained their morphologyfor approximately 2 weeks in culture. While in the short term,hPSC-derived WT1+ epicardial cells retained the epicardialcobblestone-like morphology, bFGF and TGFβ-treated cells adopted afibroid spindle or fusiform-shaped appearance typical of culturedfibroblasts and smooth muscle cells, respectively (FIGS. 6A-B). Theexpression of calponin and smooth muscle myosin heavy chain (SMMHC) inTGFβ+bFGF-induced cultures further support their smooth muscle cellidentity, and vimentin (VIM) and CD90 expression support theirfibroblast identity (FIG. 14). We also cultured WT1+TBX18+ epicardialcells in endothelial cell medium, but did not detect expression ofendothelial markers CD31 and VE-cadherin (FIG. 14). The expression ofWT1 and ZO1 significantly decreased in both bFGF and TGFβ-treatedsamples, indicating the transition from epithelial towardsmesenchymal-like cells (FIG. 6B). In addition, CDH1 (E-cadherin)expression decreased whereas CDH2 (N-cadherin) and SNAIL2 expressionincreased in all the treated samples compared to the untreated controls,suggesting bFGF and TGFβ induced EMT in hPSC-derived epicardial cells(FIGS. 6C-D).

During long term in vitro culture hPSC-derived WT1+ epicardial cellsspontaneously underwent EMT and lost WT1 expression after severalpassages, even without exogenous TGFβ or bFGF treatment (FIG. 7A). Toidentify signaling mechanisms regulating hPSC-derived epicardial cellself-renewal, we applied small molecules (Table 3) that affect pathwaysthat regulate cell proliferation to study their effects on WT1+ cellself-renewal. A83-01, an inhibitor of TGFβ signaling, enabled expansionof hPSC-derived epicardial cells that retained polarized epithelialmorphology and WT1 expression (FIGS. 7A-B). Upon A83-01 or SB431542addition, hPSC-derived epicardial cells were capable of at least 25population doublings, generating more than 10 million cells from asingle hPSC-derived epicardial cell clone (FIG. 7C). After 48 days ofexpansion, the TGFβ inhibitor-treated cells expressed significantlyhigher levels of WT1 and Ki67, a proliferative marker, than theuntreated cells (FIG. 7D). Epicardial cells generated from additionalhPSC-lines were also expandable after A83-01 treatment (FIG. 15A),presenting a cobblestone morphology and expressing high levels ofALDH1A2, WT1, ZO1 and β-catenin (FIGS. 15B-D). These findings improveour understanding of self-renewal mechanisms of the epicardium and haveimplications for generating large quantities of hPSC-derived epicardialcells for research or cell-based therapy applications.

hPSC-Derived Epicardial Cells were Similar to Primary Epicardial CellsBoth in Vitro and In Vivo

To further confirm the identity of hPSC-derived epicardial cells, RNAfrom 3 different hPSC-derived epicardial cell differentiations andprimary epicardial cells of 4 different donors were subjected to RNA-seqanalysis. FIG. 8A presents hierarchical clustering analysis of RNA-seqexpression data of hPSCs³⁴⁻³⁶, hPSC-derived endoderm (Endo)³⁴,hPSC-derived ectoderm (Ecto)³⁵, hPSC-derived mesoderm (Mes) (GSM1112833,915324, 915325), CMs³⁷, epicardial cells (Epi) derived from human stemcell lines H9, ES03, and 19-9-11, and primary epicardial cells. Thehierarchical clustering analysis showed that hPSC-derived epicardialcells were most closely related to primary epicardial cells and weredistinct from all other cell populations together as a group. Next weexplored the relationship between different cell types relevant fordevelopment including hPSC, mesoderm, cardiomyocytes, and epicardialcells, using principal component analysis (PCA) on the gene expressiondata. The 3D scores plot for the first 3 principal components shows atrend of hPSCs clustering relatively closer to mesoderm cells, fromwhich epicardial cells and CMs divergently formed (FIG. 16A).Importantly, hPSC-derived epicardial cells showed highest similaritywith donor epicardial cells. We also performed gene set enrichmentanalysis (GSEA) to identify significantly enriched pathways (p<0.05) ineach cell type in relative to hPSCs. Hierarchical clustering of theabsolute value of normalized enrichment score (NES) of these pathwaysconfirmed the similarity between epicardial cells from hPSCs and thosefrom donors (FIG. 16B). As both epicardial cells and CMs are derivedfrom mesoderm, we further compare the differences and similarities inthe enriched pathways among these cell types. We observed that while 42pathways were commonly enriched in all cell types, hPSC-derivedepicardial cells shared 36 pathways with donor epicardial cells, and 22with cardiomyocytes (FIG. 16C) (Tables 4-6). Microarray data analysishas shown the enrichment of cell adhesion and extracellular matrixorganization genes in mouse primary epicardial cells⁵³. Similarly, ourhPSC-derived or donor epicardial cells also showed enrichment inextracellular matrix related pathways and keratinocyte (epithelial)differentiation, while CMs were enriched in heart development and heartcontraction related pathways as expected. Importantly, donor epicardialcells were highly enriched in endoplasmic reticulum related pathwayscompared to hPSC-derived epicardial cells, likely indicating thematuration status of epicardial cells.

Epicardial cells can undergo EMT and give rise to cardiac fibroblastsand smooth muscle cells after transplantation into chicken embryos²⁶ orinfarcted mouse hearts²⁸. To examine the ability of hPSC-derivedepicardial cells to invade the myocardium and undergo EMT in vivo,cardiac fibroblast-derived extracellular matrix (CF-ECM) patches seededwith eGFP-labeled hPSC-derived epicardial cells (FIG. 8B) weretransferred to the heart surface (FIG. 16D) of a mouse myocardialinfarction (MI) model. eGFP+ cells were detected predominantly withinthe CF-ECM scaffold and in the epicardium beneath the scaffold beforeday 6, and scattered within the mid-myocardium after 12 days (FIG. 8Dand FIG. 16E), suggesting epicardial cells invaded the myocardium. Evenafter 12 days, the scaffold remained adherent (FIG. 16F). In addition,the hPSC-derived cells underwent EMT and differentiated intoSMA+calponin+ smooth muscle-like cells (FIG. 8D and FIG. 16G) and VIM+fibroblast-like cells in vivo. These findings demonstrate thathPSC-derived epicardial cells can invade the myocardium and form EPDCsafter infarction, underscoring their potential for cell-basedtherapeutic heart regeneration.

Discussion

Here, we report for the first time the generation of a WT1-2A-eGFPknockin stem cell line, and demonstrates efficient and robust generationof epicardial cells from multiple hPSC lines solely via stage-specificmanipulation of Wnt/β-catenin signaling under chemically-defined,albumin-free, animal component-free conditions. These hPSC-derivedepicardial cells retain many characteristics of primary epicardialcells, including formation of an epithelial sheet, expression of keyepicardial proteins WT1, TBX18, and ALDH1A2, and the ability to generatefibroblast and vascular smooth muscle lineages both in vitro and invivo. In addition, their identity was further confirmed by RNA-seqexpression data and gene set enrichment analysis (GSEA) at a globallevel. Using inducible knockdown hPSC lines, we showed that β-catenin isessential for epicardial cell induction from hPSC-derived cardiacprogenitors during the GiWiGi protocol. Given the essential roles ofβ-catenin during cardiac progenitor induction from hPSCs¹⁵, we concludethat β-catenin is required at multiple stages of hPSC differentiation toepicardial cells via small molecule modulation of canonical Wntsignaling.

This study also demonstrates long-term self-renewal of hPSC-derivedepicardial cells via TGFβ-inhibition in a chemically-defined medium. Forcell-based therapeutic applications, it is highly desirable to generatehomogeneous committed progenitors that can expand in culture anddifferentiate into various tissue-specific cells of interest, avoidingthe contamination of unwanted cell lineages, especially tumorigenichPSCs⁵⁴. We showed that inhibition of TGFβ signaling is sufficient forthe self-renewal of hPSC-derived epicardial cells, in contrast toprimary mouse epicardial cells which can self-renew in the absence of aTGFβ inhibitor⁵⁰. Recent work has demonstrated that epicardial celllineages improved the performance of the scarred myocardium bypreservation of cardiac function and attenuation of ventricularremodeling after transplantation into a MI model⁵⁵. More recently, Wanget al. identified a requirement for the epicardium of the zebrafishheart for muscle regeneration, and showed that ventricular epicardiumwas stimulated to regenerate upon Sonic hedgehog (Shh) treatment⁵⁶. Ourresults suggest that TGFβ inhibitors may impact heart regenerationfollowing injection into the epicardium in vivo, similar to strategyused to test the effect of TGFβ inhibitors on scar formation afterglaucoma surgery in rabbits⁵⁷.

In summary, our findings support a model (FIG. 8E) of human epicardialdevelopment in which small molecule-mediated exogenous modulation ofWnt/β-catenin signaling is sufficient for the specification ofepicardial cells from hPSCs. This finding is consistent with the reportthat DKK1 and DKK2 double null mice increase epicardial specificationand display a hypercellular epicardium²⁷. This completely defined,xeno-free epicardial differentiation platform can be employed toefficiently derive self-renewing epicardial cell lineages from hPSCs,which can thereby provide insights into mechanisms of heart development,maturation, and response to cardiac injury. Moreover, we show thathPSC-derived epicardial cells can invade the myocardium in an infarctedmouse model, suggesting potential applications in cell-based heartregeneration. Our results also point to TGFβ signaling as a regulator ofepicardial cell self-renewal and differentiation, indicating thepotential of TGFβ signaling modulators in heart regeneration.

TABLE 1 Antibodies Concen- Antibody Source/Isotype/clone/cat. no.tration Smooth Lab Vision/Mouse IgG2a/1A4/ms-133-p 1:100 (IS) muscleactin Cardiac Lab Vision/Mouse IgG1/13-11/ms-295-p1 1:200 troponin T(FC& IS) ISL1 DSHB/Mouse IgG2b/39.4D5-s  1:20 (IS) NKX2.5 SantaCruz/Rabbit IgG/sc-14033/H-114 1:100 (IS) Flk-1 Santa Cruz/MouseIgG1/sc-6251/A-3 1:200 (IS) Ki67 BD Biosciences/Mouse IgG1/550609 1:100(IS) WT1 Abcam/Rabbit IgG/ab89901 1:250 (FC & IS) TCF21Sigma-Aldrich/Rabbit IgG/HPA013189 1:200 (IS) TBX18 Sigma-Aldrich/RabbitIgG/HPA029014 1:200 (IS) ALDH1A2 Sigma-Aldrich/Rabbit IgG/HPA010022 1:50 (IS) ZO1 Invitrogen/Rabbit IgG/402200 1:200 (IS) β-catenin CellSignaling/Mouse IgG1/2698/L87A12 1:200 (IS) Vimentin (noSigma-Aldrich/Mouse IgG1/V6630/V9 1:200 (IS) cross reaction with mouse)VE-cadherin Santa Cruz/Mouse IgG1/F-8/sc9989 1:100 (IS) CD31ThermoFisher/Rabbit IgG/RB-10333-P 1:100 (IS) CD90 BD Pharmingen/MouseIgG1/559869 1:200 (IS) E-cadherin BD Biosciences/Mouse IgG2a/5600611:200 (IS) SMMHC Abcam/Rabbit IgG/ab82541 1:800 (IS) Calponin (noAbcam/Mouse IgG1/ab700/CALP 1:200 (IS) cross reaction with mouse) GFPDSHB/Mouse IgG1/12E6  1:20 (IS) Mitochondria Millipore/MouseIgG1/113-1/MAB1273 1:100 (IS) (human specific) β-actin CellSignaling/Rabbit mAb(HRP 1:5,000 Conjugate)/5152S/13E5 (WB) SecondaryAlexa 488 Chicken anti-Gt IgG/A-21467 1:1,000 Antibody Secondary Alexa488 Chicken anti-Rb IgG/A-21441 1:1,000 Antibody Secondary Alexa 488Goat anti-Ms IgG1/A-21121 1:1,000 Antibody Secondary Alexa 488 Goatanti-Rb IgG/A-11008 1:1,000 Antibody Secondary Alexa 594 Goat anti-MsIgG2b/A-21145 1:1,000 Antibody Secondary Alexa 594 Goat anti-RbIgG/A-11012 1:1,000 Antibody Secondary Alexa 647 Goat anti-MsIgG2b/A-21242 1:1,000 Antibody Secondary Alexa 647 Goat anti-RbIgG/A-21244 1:1,000 Antibody

TABLE 2 Oligonucleotide Primers Size (bp)/Tm Genes Sequences (5′-3′)(°C.) OCT4 F: CAGTGCCCGAAACCCACAC (SEQ ID NO: 5) 161/58 R:GGAGACCCAGCAGCCTCAAA (SEQ ID NO: 6) NANOG F:CGAAGAATAGCAATGGTGTGACG (SEQ ID NO: 7) 328/58 R:TTCCAAAGCAGCCTCCAAGTC (SEQ ID NO: 8) T F:AAGAAGGAAATGCAGCCTCA (SEQ ID NO: 9) 101/58 R:TACTGCAGGTGTGAGCAAGG (SEQ ID NO: 10) ISL1 F:CACAAGCGTCTCGGGATT (SEQ ID NO: 11) 202/58 R:AGTGGCAAGTCTTCCGACA (SEQ ID NO: 12) FLK-1 F:GTGACCAACATGGAGTCGTG (SEQ ID NO: 13) 218/60 R:TGCTTCACAGAAGACCATGC (SEQ ID NO: 14) NKX2.5 F:GCGATTATGCAGCGTCAATGAGT (SEQ ID NO: 15) 220/58 R:AACATAAATACGGGTGGGTGCGTG (SEQ ID NO: 16) TNNT2 F:TTCACCAAAGATCTGCTCCTCGCT (SEQ ID NO: 17) 165/58 R:TTATTACTGGTGTGGAGTGGGTGTGG (SEQ ID NO: 18) TBX18 F:CCCAGGACTCCCTCCTATGT (SEQ ID NO: 19) 200/59 R:TAGGAACCCTGATGGGTCTG (SEQ ID NO: 20) WT1 F:CAGCTTGAATGCATGACCTG (SEQ ID NO: 21) 200/60 R:GATGCCGACCGTACAAGAGT (SEQ ID NO: 22) TCF21 F:ACCCTCTTCCTCGCTTTCTC (SEQ ID NO: 23) 180/59 R:TGCTCTCGTTGGAAGTCACA (SEQ ID NO: 24) ALDH1A2 F:CTCCTCTGTCACACCCCATT (SEQ ID NO: 25) 198/59 R:TTGACAGCTGGAAAGATGGA (SEQ ID NO: 26) SNA12 F:ACAGAGCATTTGCAGACAGG (SEQ ID NO: 27) 147/59 R:GTGCTACACAGCAGCCAGAT (SEQ ID NO: 28) CDH1 F:TTCTGCTGCTCTTGCTGTTT (SEQ ID NO: 29) 142/59 R:TGGCTCAAGTCAAAGTCCTG (SEQ ID NO: 30) CDH2 F:CTCCAATCAACTTGCCAGAA (SEQ ID NO: 31) 136/58 R:ATACCAGTTGGAGGCTGGTC (SEQ ID NO: 32) CTNNB1 F:GAATGAGACTGCTGATCTTGGAC (SEQ ID NO: 33) 250/58 R:CTGATTGCTGTCACCTGGAG (SEQ ID NO: 34) GAPDH F:GTGGACCTGACCTGCCGTCT (SEQ ID NO: 35) 152/58 R:GGAGGAGTGGGTGTCGCTGT (SEQ ID NO: 36) WT1 K1 F:GGTCTTGGTTTCTGCTGGAC (SEQ ID NO: 37) 2777/60 (Red) R:AAGTCGTGCTGCTTCATGTG (SEQ ID NO: 38) WT1 K1 F:TGAAAAGCCCTTCAGCTGTC (SEQ ID NO: 39) 204 or 2847/60 (Blue) R:TGAGGAGGAGTGGAGAGTCAG (SEQ ID NO: 40)

TABLE 3 Signaling Modulators Targeted Concen- Modulator pathway trationbFGF FGF 10 ng/mL BMP4 BMP 10 ng/mL Dorsomorphin (DM) BMP 4 μM IWP2 Wnt5 μM CHIR99021 (CHIR) Wnt 1~9 μM CHIR98014 Wnt 0.3 μM BIO-acetoxime Wnt0.3 μM Purmorphamine (PURM) Hedgehog 2 μM Retinoic acid (RA) RA 2 μMPD0325901 (PD) MEK 0.5 μM Verteporfin (VP) Hippo pathway 1 μM RO4929097(RO) Notch 2 μM TGFβ1 TGFβ 5 ng/mL A83-01 TGFβ 0.5 μM SB431542 TGFβ 2 μM

TABLE 4 Top 15 Gene Annotations enriched in donor epicardial cellscompared to hPSCs GO Description NES p-value Positive regulation ofI-kappaB kinase NF 2.068 0 kappaB cascade Regulation of I-kappaB NFkappaB cascade 1.983 0 Endoplasmic reticulum part 1.900 0 Endoplasmicreticulum membrane 1.810 0 I-kappaB kinase NF kappaB cascade 1.804 0Intrinsic to organelle membrane 1.803 0 Positive regulation of signaltransduction 1.800 0 ER to golgi vesicle mediated transport 1.787 0.009Extracellular matrix part 1.782 0 Keratinocyte differentiation 1.7680.020 Hematopoietin interferon class D200 domain 1.765 0 cytokinereceptor binding Integral to endoplasmic reticulum membrane 1.765 0.016ER golgi intermediate compartment 1.763 0.005 Integral to organellemembrane 1.759 0 * NES: normalized enrichment score. P-value of 0 means<0.0001.

TABLE 5 Top 15 Gene Annotations enriched in hPSC- derived epicardialcells compared to hPSCs Number of GO Description genes p-value Sulfuricester hydrolase activity 1.776 0.004 Extracellular matrix 1.760 0Proteinaceous extracellular matrix 1.751 0 Extracellular matrix part1.727 0.002 Muscle development 1.709 0 Negative regulation of cell cycle1.695 0.002 Collagen 1.668 0.0052 Cell cycle arrest GO 0007050 1.6620.0032 Muscle cell differentiation 1.649 0.011 Skeletal development1.637 0 Skeletal muscle development 1.604 0.008 Keratinocytedifferentiation 1.600 0.009 Myoblast differentiation 1.594 0.010Striated muscle development 1.576 0.010 * NES: normalized enrichmentscore. P-value of 0 means <0.0001.

TABLE 6 Top 15 Gene Annotations enriched in hPSC-derived cardiomyocytes(CMs) compared to hPSCs GO Description NES p-value Regulation of heartcontraction 2.070 0.000 Structural constituent of muscle 1.971 0.000Muscle development 1.890 0.000 Mitochondrial membrane part 1.842 0.000Myosin complex 1.832 0.000 Growth factor activity 1.819 0.002Mitochondrial membrane 1.817 0.000 Energy derivation by oxidation oforganic compounds 1.803 0.000 Heart development 1.781 0.000Mitochondrial inner membrane 1.774 0.000 Mitochondrial respiratory chain1.758 0.000 Mitochondrial envelope 1.739 0.000 Contractile fiber part1.696 0.002 Generation of precursor metabolites and energy 1.665 0.000 *NES: normalized enrichment score. P-value of 0 means <0.0001.

REFERENCES

All publications, including but not limited to patents and patentapplications, cited below are herein incorporated by reference as thoughset forth in their entirety in the present application.

-   1. Brade, T., Pane, L. S., Moretti, A., Chien, K. R. & Laugwitz,    K.-L. Embryonic heart progenitors and cardiogenesis. Cold Spring    Harb. Perspect. Med. 3, a013847 (2013).-   2. Männer, J. & Ruiz-Lozano, P. Development and Function of the    Epicardium. Advances in Developmental Biology 18, 333-357 (2007).-   3. Riley, P. R. An Epicardial Floor Plan for Building and Rebuilding    the Mammalian Heart. Curr. Top. Dev. Biol. 100, 233-251 (2012).-   4. Pérez-Pomares, J.-M. et al. Origin of coronary endothelial cells    from epicardial mesothelium in avian embryos. Int. J. Dev. Biol. 46,    1005-13 (2002).-   5. Smart, N. et al. Thymosin beta4 induces adult epicardial    progenitor mobilization and neovascularization. Nature 445, 177-82    (2007).-   6. Zhou, B. et al. Adult mouse epicardium modulates myocardial    injury by secreting paracrine factors. J. Clin. Invest. 121,    1894-1904 (2011).-   7. Zhou, B. & Pu, W. T. Epicardial epithelial to mesenchymal    transition in injured heart. J. Cell. Mol. Med. 15, 2781-2783    (2012).-   8. Kikuchi, K. et al. Retinoic Acid Production by Endocardium and    Epicardium Is an Injury Response Essential for Zebrafish Heart    Regeneration. Dev. Cell 20, 397-404 (2011).-   9. Lepilina, A. et al. A Dynamic Epicardial Injury Response Supports    Progenitor Cell Activity during Zebrafish Heart Regeneration. Cell    127, 607-619 (2006).-   10. Zhou, B. et al. Epicardial progenitors contribute to the    cardiomyocyte lineage in the developing heart. Nature 454, 109-13    (2008).-   11. Thomson, J. A. Embryonic Stem Cell Lines Derived from Human    Blastocysts. Science 282, 1145-1147 (1998).-   12. Murry, C. E. & Keller, G. Differentiation of embryonic stem    cells to clinically relevant populations: lessons from embryonic    development. Cell 132, 661-80 (2008).-   13. Ashton, R. S., Keung, A. J., Peltier, J. & Schaffer, D. V.    Progress and prospects for stem cell engineering. Annu. Rev. Chem.    Biomol. Eng. 2, 479-502 (2011).-   14. Kattman, S. J. et al. Stage-specific optimization of    activin/nodal and BMP signaling promotes cardiac differentiation of    mouse and human pluripotent stem cell lines. Cell Stem Cell 8,    228-40 (2011).-   15. Lian, X. J. et al. Robust cardiomyocyte differentiation from    human pluripotent stem cells via temporal modulation of canonical    Wnt signaling. Proc. Natl. Acad. Sci. U.S.A. 109, E1848-E1857    (2012).-   16. Lian, X. et al. Directed cardiomyocyte differentiation from    human pluripotent stem cells by modulating Wnt/β-catenin signaling    under fully defined conditions. Nat. Protoc. 8, 162-75 (2013).-   17. Minami, I. et al. A small molecule that promotes cardiac    differentiation of human pluripotent stem cells under defined,    cytokine- and xeno-free conditions. Cell Rep. 2, 1448-60 (2012).-   18. Bao, X. et al. Chemically-defined albumin-free differentiation    of human pluripotent stem cells to endothelial progenitor cells.    Stem Cell Res. 15, 122-129 (2015).-   19. Lian, X. et al. Efficient Differentiation of Human Pluripotent    Stem Cells to Endothelial Progenitors via Small-Molecule Activation    of WNT Signaling. Stem Cell Reports 3, 804-16 (2014).-   20. Sahara, M. et al. Manipulation of a VEGF-Notch signaling circuit    drives formation of functional vascular endothelial progenitors from    human pluripotent stem cells. Cell Res. 24, 820-41 (2014).-   21. Samuel, R. et al. Generation of functionally competent and    durable engineered blood vessels from human induced pluripotent stem    cells. Proc. Natl. Acad. Sci. U.S.A. 110, 12774-9 (2013).-   22. Wang, A. et al. Derivation of Smooth Muscle Cells with Neural    Crest Origin from Human Induced Pluripotent Stem Cells. Cells    Tissues Organs 195, 5-14 (2012).-   23. Wang, Y. et al. Engineering vascular tissue with functional    smooth muscle cells derived from human iPS cells and nanofibrous    scaffolds. Biomaterials 35, 8960-8969 (2014).-   24. Cheung, C., Bernardo, A. S., Trotter, M. W. B., Pedersen, R. A.    & Sinha, S. Generation of human vascular smooth muscle subtypes    provides insight into embryological origin-dependent disease    susceptibility. Nature Biotechnology 30, 165-173 (2012).-   25. Witty, A. D. et al. Generation of the epicardial lineage from    human pluripotent stem cells. Nat. Biotechnol. 32, 1026-1035 (2014).-   26. Iyer, D. et al. Robust derivation of epicardium and its    differentiated smooth muscle cell progeny from human pluripotent    stem cells. Development 142, 1528-1541 (2015).-   27. Phillips, M. D., Mukhopadhyay, M., Poscablo, C. & Westphal, H.    Dkk1 and Dkk2 regulate epicardial specification during mouse heart    development. Int. J. Cardiol. 150, 186-92 (2011).-   28. van Tuyn, J. et al. Epicardial cells of human adults can undergo    an epithelial-to-mesenchymal transition and obtain characteristics    of smooth muscle cells in vitro. Stem Cells 25, 271-278 (2007).-   29. Hockemeyer, D. et al. Genetic engineering of human pluripotent    cells using TALE nucleases. Nat. Biotechnol. 29, 731-4 (2011).-   30. Lian, X. et al. A small molecule inhibitor of SRC family kinases    promotes simple epithelial differentiation of human pluripotent stem    cells. PLoS One 8, e60016 (2013).-   31. Schmuck, E. G. et al. Cardiac fibroblast-derived 3D    extracellular matrix seeded with mesenchymal stem cells as a novel    device to transfer cells to the ischemic myocardium. Cardiovasc.    Eng. Technol. 5, 119-131 (2014).-   32. Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced    aligner with low memory requirements. Nat. Methods 12, 357-360    (2015).-   33. Ramsköld, D., Wang, E. T., Burge, C. B. & Sandberg, R. An    abundance of ubiquitously expressed genes revealed by tissue    transcriptome sequence data. PLoS Comput. Biol. 5, e1000598 (2009).-   34. Dye, B. R. et al. In vitro generation of human pluripotent stem    cell derived lung organoids. Elife 4, e05098 (2015).-   35. Tadeu, A. M. B. et al. Transcriptional profiling of ectoderm    specification to keratinocyte fate in human embryonic stem cells.    PLoS One 10, e0122493 (2015).-   36. Prasain, N. et al. Differentiation of human pluripotent stem    cells to cells similar to cord-blood endothelial colony-forming    cells. Nat. Biotechnol. 32, 1151-7 (2014).-   37. Palpant, N. J. et al. Inhibition of β-catenin signaling    respecifies anterior-like endothelium into beating human    cardiomyocytes. Development 142, 3198-209 (2015).-   38. Subramanian, A. et al. Gene set enrichment analysis: a    knowledge-based approach for interpreting genome-wide expression    profiles. Proc. Natl. Acad. Sci. U.S.A. 102, 15545-50 (2005).-   39. Lian, X. et al. Chemically defined, albumin-free human    cardiomyocyte generation. Nat. Methods 12, 595-596 (2015).-   40. Nakanishi, M. et al. Directed induction of anterior and    posterior primitive streak by Wnt from embryonic stem cells cultured    in a chemically defined serum-free medium. FASEB J. 23, 114-22    (2009).-   41. Zhou, B., von Gise, A., Ma, Q., Rivera-Feliciano, J. & Pu, W. T.    Nkx2-5- and Isl1-expressing cardiac progenitors contribute to    proepicardium. Biochem. Biophys. Res. Commun. 375, 450-3 (2008).-   42. Moore, A. W., McInnes, L., Kreidberg, J., Hastie, N. D. &    Schedl, A. YAC complementation shows a requirement for Wt1 in the    development of epicardium, adrenal gland and throughout    nephrogenesis. Development 126, 1845-57 (1999).-   43. Martínez-Estrada, O. M. et al. Wt1 is required for    cardiovascular progenitor cell formation through transcriptional    control of Snail and E-cadherin. Nat. Genet. 42, 89-93 (2010).-   44. Hockemeyer, D. et al. Genetic engineering of human pluripotent    cells using TALE nucleases. Nat. Biotechnol. 29, 731-4 (2011).-   45. Kofidis, T. et al. Insulin-like growth factor promotes    engraftment, differentiation, and functional improvement after    transfer of embryonic stem cells for myocardial restoration. Stem    Cells 22, 1239-45 (2004).-   46. Engels, M. C. et al. Insulin-like growth factor promotes cardiac    lineage induction in vitro by selective expansion of early mesoderm.    Stem Cells 32, 1493-502 (2014).-   47. Cao, N. et al. Ascorbic acid enhances the cardiac    differentiation of induced pluripotent stem cells through promoting    the proliferation of cardiac progenitor cells. Cell Res. 22, 219-36    (2012).-   48. Ueno, S. et al. Biphasic role for Wnt/beta-catenin signaling in    cardiac specification in zebrafish and embryonic stem cells. Proc.    Natl. Acad. Sci. U.S.A. 104, 9685-90 (2007).-   49. David, R. et al. MesP1 drives vertebrate cardiovascular    differentiation through Dkk-1-mediated blockade of Wnt-signalling.    Nat. Cell Biol. 10, 338-45 (2008).-   50. Ruiz-Villalba, A., Ziogas, A., Ehrbar, M. & Pérez-Pomares, J. M.    Characterization of epicardial-derived cardiac interstitial cells:    differentiation and mobilization of heart fibroblast progenitors.    PLoS One 8, e53694 (2013).-   51. Pérez-Pomares, J. M. et al. Experimental studies on the    spatiotemporal expression of WT1 and RALDH2 in the embryonic avian    heart: a model for the regulation of myocardial and valvuloseptal    development by epicardially derived cells (EPDCs). Dev. Biol. 247,    307-26 (2002).-   52. Garriock, R. J., Mikawa, T. & Yamaguchi, T. P. Isolation and    culture of mouse proepicardium using serum-free conditions. Methods    66, 365-9 (2014).-   53. Bochmann, L. et al. Revealing new mouse epicardial cell markers    through transcriptomics. PLoS One 5, e11429 (2010).-   54. Lam, J. T., Moretti, A. & Laugwitz, K.-L. Multipotent progenitor    cells in regenerative cardiovascular medicine. Pediatr. Cardiol. 30,    690-8 (2009).-   55. Winter, E. M. et al. Preservation of left ventricular function    and attenuation of remodeling after transplantation of human    epicardium-derived cells into the infarcted mouse heart. Circulation    116, 917-27 (2007).-   56. Wang, J., Cao, J., Dickson, A. L. & Poss, K. D. Epicardial    regeneration is guided by cardiac outflow tract and Hedgehog    signalling. Nature 522, 226-230 (2015).-   57. Xiao, Y., Liu, K., Shen, J., Xu, G. & Ye, W. SB-431542    inhibition of scar formation after filtration surgery and its    potential mechanism. Invest. Ophthalmol. Vis. Sci. 50, 1698-706    (2009).

The present invention has been presented by way of illustration and isnot intended to be limited to the disclosed embodiments. Accordingly,those skilled in the art will realize that the invention is intended toencompass all modifications and alternative arrangements within thespirit and scope of the invention as set forth in the appended claims.

We claim:
 1. A method for generating a population of epicardial cellsfrom human pluripotent stem cells, the method comprising culturing humancardiac progenitor cells, differentiated from human pluripotent stemcells, in a chemically defined, albumin-free culture medium thatcomprises an activator of Wnt/β-catenin signaling and that does notcomprise Bone Morphogenetic Protein 4 (BMP4), whereby a cell populationcomprising human epicardial cells is obtained.
 2. The method of claim 1,wherein the human cardiac progenitor cells express one or more of Isl1,Nkx2.5, and Flk-1.
 3. The method of claim 1, wherein at least 95% ofcells of the cell population are epicardial cells positive forexpression of Wilms' tumor suppressor protein (WT1).
 4. The method ofclaim 3, wherein the epicardial cells are self-renewing for at least 25population doublings when cultured in the presence of an inhibitor ofTGFβ signaling.
 5. The method of claim 1, wherein the activator ofWnt/β-catenin signaling is a Gsk3 inhibitor.
 6. The method of claim 5,wherein the Gsk3 inhibitor is a small molecule selected from the groupconsisting of CHIR99021, CHIR98014, BIO-acetoxime, BIO, LiCl, SB216763,SB415286, AR A014418, 1-Azakenpaullone, and Bis-7-indolylmaleimide. 7.The method of claim 5, wherein the Gsk3 inhibitor is CHIR99021 and ispresent in a concentration of about 0.2 μM to about 9 μM.
 8. The methodof claim 1, wherein the human cardiac progenitor cells are obtained by amethod comprising (i) culturing human pluripotent stem cells in achemically defined, albumin-free culture medium comprising an activatorof Wnt/β-catenin signaling to obtain a first cell population comprisingmesodermal cells positive for expression of Brachyury/T; and (ii)culturing the first cell population in a chemically defined culturemedium that comprises an inhibitor of Wnt/β-catenin signaling, whereby acell population comprising human cardiac progenitor cells is obtained.9. The method of claim 8, wherein the human cardiac progenitor cellsexpress one or more of Isl1, Nkx2.5, and Flk-1.
 10. The method of claim8, wherein the activator of Wnt/β-catenin signaling is a Gsk3 inhibitor.11. The method of claim 10, wherein the Gsk3 inhibitor is a smallmolecule selected from the group consisting of CHIR99021, CHIR98014,BIO-acetoxime, BIO, LiCl, SB216763, SB415286, AR A014418,1-Azakenpaullone, and Bis-7-indolylmaleimide.
 12. The method of claim 8,wherein the inhibitor of Wnt/β-catenin signaling is selected from thegroup consisting of a small molecule that stabilizes axin and stimulatesβ-catenin degradation, an inhibitor of porcupine, an antibody thatblocks activation of a Wnt ligand receptor, an antibody that binds toone or more Wnt ligand family members, and a short hairpin interferingRNA (shRNA) for β-catenin in the first cell population.
 13. The methodof claim 12, wherein the small molecule that stimulates β-catenindegradation and stabilizes axin is XAV939.
 14. The method of claim 12,wherein the porcupine inhibitor is selected from the group consisting ofIWP2 and IWP4, or a combination thereof.
 15. The method of claim 12,wherein the porcupine inhibitor is present in a concentration of about 1μM to about 4 μM.
 16. The method of claim 1, wherein no cell separationor selection step is used to obtain the cell population comprisingepicardial cells.
 17. A method for long-term in vitro maintenance ofself-renewing human epicardial cells, the method comprising: culturinghuman cardiac progenitor cells differentiated from human pluripotentstem cells in a chemically defined, albumin-free culture medium thatcomprises an activator of Wnt/β-catenin signaling and that does notcomprise Bone Morphogenetic Protein 4 (BMP4), whereby a cell populationcomprising human epicardial cells is obtained; and culturing the cellpopulation comprising human epicardial cells in the presence of aninhibitor of TGFβ signaling, whereby the human epicardial cells aremaintained in vitro as self-renewing epicardial cells for at least 25population doublings, are not immortalized, and maintain the ability toundergo epithelial-to-mesenchymal transition (EMT).